Cellobiohydrolase variants and polynucleotides encoding same

ABSTRACT

The present invention relates to variants of a parent cellobiohydrolase II. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 15/446,676, filed Mar. 1, 2017, which is a divisional application ofU.S. application Ser. No. 14/513,087, filed Oct. 13, 2014, now U.S. Pat.No. 9,611,463, which is a divisional application of U.S. applicationSer. No. 14/034,209, filed Sep. 23, 2013, now U.S. Pat. No. 8,859,253,which is a divisional application of U.S. application Ser. No.12/908,339, filed Oct. 20, 2010, now U.S. Pat. No. 8,541,651, whichclaims the benefit of U.S. Provisional Application Ser. No. 61/254,408,filed Oct. 23, 2009. The contents of these applications are fulllyincorporated herein by reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with Government support under CooperativeAgreement DE-FC36-08G018080 awarded by the Department of Energy. Thegovernment has certain rights in this invention.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to variants of a cellobiohydrolase II,polynucleotides encoding the variants, methods of producing thevariants, and methods of using the variants.

Description of the Related Art

Cellulose is a polymer of the simple sugar glucose covalently linked bybeta-1,4-bonds. Many microorganisms produce enzymes that hydrolyzebeta-linked glucans. These enzymes include endoglucanases,cellobiohydrolases, and beta-glucosidases. Endoglucanases digest thecellulose polymer at random locations, opening it to attack bycellobiohydrolases. Cellobiohydrolases sequentially release molecules ofcellobiose from the ends of the cellulose polymer. Cellobiose is awater-soluble beta-1,4-linked dimer of glucose. Beta-glucosidaseshydrolyze cellobiose to glucose.

The conversion of lignocellulosic feedstocks into ethanol has theadvantages of the ready availability of large amounts of feedstock, thedesirability of avoiding burning or land filling the materials, and thecleanliness of the ethanol fuel. Wood, agricultural residues, herbaceouscrops, and municipal solid wastes have been considered as feedstocks forethanol production. These materials primarily consist of cellulose,hemicellulose, and lignin. Once the lignocellulose is converted tofermentable sugars, e.g., glucose, the fermentable sugars are easilyfermented by yeast into ethanol.

WO 2006/074005 discloses variants of a Hypocrea jecorinacellobiohydrolase II. Heinzelman et al., 2009, Proceedings of theNational Academy of Sciences USA 106:5610-5615 discloses a family ofthermostable fungal cellulases created by structure-guidedrecombination. Heinzelman et al., 2009, Journal of Biological Chemistry284, 26229-26233 discloses a single mutation that contributes tostability of a fungal cellulase.

It would be advantageous in the art to improve the ability ofpolypeptides having cellobiohydrolase activity to improve enzymaticdegradation of lignocellulosic feedstocks.

The present invention provides variants of a parent cellobiohydrolase IIwith increased thermostability compared to its parent.

SUMMARY OF THE INVENTION

The present invention relates to isolated variants of a parentcellobiohydrolase II, comprising a substitution at one or more (several)positions corresponding to positions 272, 287, 325, 347, 357, 363, 409,464, and 476 of the mature polypeptide of SEQ ID NO: 2, wherein thevariants have cellobiohydrolase II activity. In one aspect, the isolatedvariants further comprise a substitution at a position corresponding toposition 435 of the mature polypeptide of SEQ ID NO: 2.

The present invention also relates to isolated polynucleotides encodingthe variants; nucleic acid constructs, vectors, and host cellscomprising the polynucleotides; and methods of producing the variants.

The present invention also relates to methods for degrading orconverting a cellulosic material, comprising: treating the cellulosicmaterial with an enzyme composition in the presence of a variant havingcellobiohydrolase II activity of the present invention. In one aspect,the method further comprises recovering the degraded or convertedcellulosic material.

The present invention also relates to methods of producing afermentation product, comprising: (a) saccharifying a cellulosicmaterial with an enzyme composition in the presence of a variant havingcellobiohydrolase II activity of the present invention; (b) fermentingthe saccharified cellulosic material with one or more (several)fermenting microorganisms to produce the fermentation product; and (c)recovering the fermentation product from the fermentation.

The present invention also relates to methods of fermenting a cellulosicmaterial, comprising: fermenting the cellulosic material with one ormore (several) fermenting microorganisms, wherein the cellulosicmaterial is saccharified with an enzyme composition in the presence of avariant having cellobiohydrolase II activity of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a comparison of the residual activity for wild-typeThielavia terrestris Family GH6A cellobiohydrolase II and severalvariants of the Thielavia terrestris Family GH6A cellobiohydrolase II in100 mM NaCl-50 mM sodium acetate pH 5.0 for 20 minutes at 67° C.

DEFINITIONS

Cellobiohydrolase: The term “cellobiohydrolase” means a1,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91), which catalyzes thehydrolysis of 1,4-beta-D-glucosidic linkages in cellulose,cellooligosaccharides, or any beta-1,4-linked glucose containingpolymer, releasing cellobiose from the reducing or non-reducing ends ofthe chain (Teeri, 1997, Crystalline cellulose degradation: New insightinto the function of cellobiohydrolases, Trends in Biotechnology 15:160-167; Teeri et al., 1998, Trichoderma reesei cellobiohydrolases: whyso efficient on crystalline cellulose?, Biochem. Soc. Trans. 26:173-178). For purposes of the present invention, cellobiohydrolaseactivity is determined according to the procedures described by Lever etal., 1972, Anal. Biochem. 47: 273-279; van Tilbeurgh et al., 1982, FEBSLetters, 149: 152-156; van Tilbeurgh and Claeyssens, 1985, FEBS Letters,187: 283-288; and Tomme et al., 1988, Eur. J. Biochem. 170: 575-581; andvan Tilbeurgh et al., 1985, Eur. J. Biochem. 148: 329-334. The Lever etal. method can be employed to assess hydrolysis of cellulose in cornstover, while the methods of van Tilbeurgh et al. and Tomme et al. canbe used to determine cellobiohydrolase I activity on4-methylumbelliferyl-β-D-lactopyranoside. In the present invention, theassay described in Example 5 can be used to measure cellobiohydrolase IIactivity.

Variant: The term “variant” means a cellobiohydrolase II comprising analteration, i.e., a substitution, insertion, and/or deletion, at one ormore (several) positions. A substitution means a replacement of an aminoacid occupying a position with a different amino acid; a deletion meansremoval of an amino acid occupying a position; and an insertion meansadding 1-5 amino acids adjacent to an amino acid occupying a position.

Mutant: The term “mutant” means a polynucleotide encoding a variant.

Wild-type cellobiohydrolase II: The term “wild-type cellobiohydrolaseII” means a cellobiohydrolase II expressed by a naturally occurringmicroorganism, such as a bacterium, yeast, or filamentous fungus foundin nature.

Parent or parent cellobiohydrolase II: The term “parent” or “parentcellobiohydrolase II” means a cellobiohydrolase II to which analteration is made to produce the enzyme variants of the presentinvention. The parent may be a naturally occurring (wild-type)polypeptide or a variant thereof.

Isolated or purified: The terms “isolated” and “purified” mean apolypeptide or polynucleotide that is removed from at least onecomponent with which it is naturally associated. For example, a variantmay be at least 1% pure, e.g., at least 5% pure, at least 10% pure, atleast 20% pure, at least 40% pure, at least 60% pure, at least 80% pure,at least 90% pure, or at least 95% pure, as determined by SDS-PAGE and apolynucleotide may be at least 1% pure, e.g., at least 5% pure, at least10% pure, at least 20% pure, at least 40% pure, at least 60% pure, atleast 80% pure, at least 90% pure, or at least 95% pure, as determinedby agarose electrophoresis.

Cellulolytic enzyme or cellulase: The term “cellulolytic enzyme” or“cellulase” means one or more (several) enzymes that hydrolyze acellulosic material. Such enzymes include endoglucanase(s),cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof. Thetwo basic approaches for measuring cellulolytic activity include: (1)measuring the total cellulolytic activity, and (2) measuring theindividual cellulolytic activities (endoglucanases, cellobiohydrolases,and beta-glucosidases) as reviewed in Zhang et al., Outlook forcellulase improvement: Screening and selection strategies, 2006,Biotechnology Advances 24: 452-481. Total cellulolytic activity isusually measured using insoluble substrates, including Whatman N21filter paper, microcrystalline cellulose, bacterial cellulose, algalcellulose, cotton, pretreated lignocellulose, etc. The most common totalcellulolytic activity assay is the filter paper assay using Whatman Nº1filter paper as the substrate. The assay was established by theInternational Union of Pure and Applied Chemistry (IUPAC) (Ghose, 1987,Measurement of cellulase activities, Pure Appl. Chem. 59: 257-68).

For purposes of the present invention, cellulolytic enzyme activity isdetermined by measuring the increase in hydrolysis of a cellulosicmaterial by cellulolytic enzyme(s) under the following conditions: 1-20mg of cellulolytic enzyme protein/g of cellulose in PCS for 3-7 days at50° C. compared to a control hydrolysis without addition of cellulolyticenzyme protein. Typical conditions are 1 ml reactions, washed orunwashed PCS, 5% insoluble solids, 50 mM sodium acetate pH 5, 1 mMMnSO₄, 50° C., 72 hours, sugar analysis by AMINEX® HPX-87H column(Bio-Rad Laboratories, Inc., Hercules, Calif., USA).

Endoglucanase: The term “endoglucanase” means anendo-1,4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase (E.C. 3.2.1.4),which catalyzes endohydrolysis of 1,4-beta-D-glycosidic linkages incellulose, cellulose derivatives (such as carboxymethyl cellulose andhydroxyethyl cellulose), lichenin, beta-1,4 bonds in mixed beta-1,3glucans such as cereal beta-D-glucans or xyloglucans, and other plantmaterial containing cellulosic components. Endoglucanase activity can bedetermined by measuring reduction in substrate viscosity or increase inreducing ends determined by a reducing sugar assay (Zhang et al., 2006,Biotechnology Advances 24: 452-481). For purposes of the presentinvention, endoglucanase activity is determined using carboxymethylcellulose (CMC) as substrate according to the procedure of Ghose, 1987,Pure and Appl. Chem. 59: 257-268, at pH 5, 40° C.

Beta-glucosidase: The term “beta-glucosidase” means a beta-D-glucosideglucohydrolase (E.C. 3.2.1.21), which catalyzes the hydrolysis ofterminal non-reducing beta-D-glucose residues with the release ofbeta-D-glucose. For purposes of the present invention, beta-glucosidaseactivity is determined according to the basic procedure described byVenturi et al., 2002, Extracellular beta-D-glucosidase from Chaetomiumthermophilum var. coprophilum: production, purification and somebiochemical properties, J. Basic Microbiol. 42: 55-66. One unit ofbeta-glucosidase is defined as 1.0 μmole of p-nitrophenolate anionproduced per minute at 25° C., pH 4.8 from 1 mMp-nitrophenyl-beta-D-glucopyranoside as substrate in 50 mM sodiumcitrate containing 0.01% TWEEN® 20.

Polypeptide having cellulolytic enhancing activity: The term“polypeptide having cellulolytic enhancing activity” means a GH61polypeptide that enhances the hydrolysis of a cellulosic material byenzyme having cellulolytic activity. For purposes of the presentinvention, cellulolytic enhancing activity is determined by measuringthe increase in reducing sugars or the increase of the total ofcellobiose and glucose from the hydrolysis of a cellulosic material bycellulolytic enzyme under the following conditions: 1-50 mg of totalprotein/g of cellulose in PCS, wherein total protein is comprised of50-99.5% w/w cellulolytic enzyme protein and 0.5-50% w/w protein of aGH61 polypeptide having cellulolytic enhancing activity for 1-7 days at50° C. compared to a control hydrolysis with equal total protein loadingwithout cellulolytic enhancing activity (1-50 mg of cellulolyticprotein/g of cellulose in PCS). In a preferred aspect, a mixture ofCELLUCLAST® 1.5 L (Novozymes A/S, Bagsvrd, Denmark) in the presence of2-3% of total protein weight Aspergillus oryzae beta-glucosidase(recombinantly produced in Aspergillus oryzae according to WO 02/095014)or 2-3% of total protein weight Aspergillus fumigatus beta-glucosidase(recombinantly produced in Aspergillus oryzae as described in WO2002/095014) of cellulase protein loading is used as the source of thecellulolytic activity.

The GH61 polypeptides having cellulolytic enhancing activity enhance thehydrolysis of a cellulosic material catalyzed by enzyme havingcellulolytic activity by reducing the amount of cellulolytic enzymerequired to reach the same degree of hydrolysis preferably at least1.01-fold, more preferably at least 1.05-fold, more preferably at least1.10-fold, more preferably at least 1.25-fold, more preferably at least1.5-fold, more preferably at least 2-fold, more preferably at least3-fold, more preferably at least 4-fold, more preferably at least5-fold, even more preferably at least 10-fold, and most preferably atleast 20-fold.

Family 61 glycoside hydrolase: The term “Family 61 glycoside hydrolase”or “Family GH61” or “GH61” means a polypeptide falling into theglycoside hydrolase Family 61 according to Henrissat B., 1991, Aclassification of glycosyl hydrolases based on amino-acid sequencesimilarities, Biochem. J. 280: 309-316, and Henrissat B., and BairochA., 1996, Updating the sequence-based classification of glycosylhydrolases, Biochem. J. 316: 695-696.

Hemicellulolytic enzyme or hemicellulase: The term “hemicellulolyticenzyme” or “hemicellulase” means one or more (several) enzymes thathydrolyze a hemicellulosic material. See, for example, Shallom, D. andShoham, Y. Microbial hemicellulases. Current Opinion In Microbiology,2003, 6(3): 219-228). Hemicellulases are key components in thedegradation of plant biomass. Examples of hemicellulases include, butare not limited to, an acetylmannan esterase, an acetyxylan esterase, anarabinanase, an arabinofuranosidase, a coumaric acid esterase, aferuloyl esterase, a galactosidase, a glucuronidase, a glucuronoylesterase, a mannanase, a mannosidase, a xylanase, and a xylosidase. Thesubstrates of these enzymes, the hemicelluloses, are a heterogeneousgroup of branched and linear polysaccharides that are bound via hydrogenbonds to the cellulose microfibrils in the plant cell wall, crosslinkingthem into a robust network. Hemicelluloses are also covalently attachedto lignin, forming together with cellulose a highly complex structure.The variable structure and organization of hemicelluloses require theconcerted action of many enzymes for its complete degradation. Thecatalytic modules of hemicellulases are either glycoside hydrolases(GHs) that hydrolyze glycosidic bonds, or carbohydrate esterases (CEs),which hydrolyze ester linkages of acetate or ferulic acid side groups.These catalytic modules, based on homology of their primary sequence,can be assigned into GH and CE families marked by numbers. Somefamilies, with overall similar fold, can be further grouped into clans,marked alphabetically (e.g., GH-A). A most informative and updatedclassification of these and other carbohydrate active enzymes isavailable on the Carbohydrate-Active Enzymes (CAZy) database.Hemicellulolytic enzyme activities can be measured according to Ghoseand Bisaria, 1987, Pure & Appl. Chem. 59: 1739-1752.

Xylan degrading activity or xylanolytic activity: The term “xylandegrading activity” or “xylanolytic activity” means a biologicalactivity that hydrolyzes xylan-containing material. The two basicapproaches for measuring xylanolytic activity include: (1) measuring thetotal xylanolytic activity, and (2) measuring the individual xylanolyticactivities (e.g., endoxylanases, beta-xylosidases, arabinofuranosidases,alpha-glucuronidases, acetylxylan esterases, feruloyl esterases, andalpha-glucuronyl esterases). Recent progress in assays of xylanolyticenzymes is summarized in several publications including Biely andPuchard, Recent progress in the assays of xylanolytic enzymes, 2006,Journal of the Science of Food and Agriculture 86(11): 1636-1647;Spanikova and Biely, 2006, Glucuronoyl esterase—Novel carbohydrateesterase produced by Schizophyllum commune, FEBS Letters 580(19):4597-4601; Herrmann, Vrsanska, Jurickova, Hirsch, Biely, and Kubicek,1997, The beta-D-xylosidase of Trichoderma reesei is a multifunctionalbeta-D-xylan xylohydrolase, Biochemical Journal 321: 375-381.

Total xylan degrading activity can be measured by determining thereducing sugars formed from various types of xylan, including, forexample, oat spelt, beechwood, and larchwood xylans, or by photometricdetermination of dyed xylan fragments released from various covalentlydyed xylans. The most common total xylanolytic activity assay is basedon production of reducing sugars from polymeric 4-O-methylglucuronoxylan as described in Bailey, Biely, Poutanen, 1992,Interlaboratory testing of methods for assay of xylanase activity,Journal of Biotechnology 23(3): 257-270. Xylanase activity can also bedetermined with 0.2% AZCL-arabinoxylan as substrate in 0.01% TRITON®X-100 and 200 mM sodium phosphate buffer pH 6 at 37° C. One unit ofxylanase activity is defined as 1.0 μmole of azurine produced per minuteat 37° C., pH 6 from 0.2% AZCL-arabinoxylan as substrate in 200 mMsodium phosphate pH 6 buffer.

For purposes of the present invention, xylan degrading activity isdetermined by measuring the increase in hydrolysis of birchwood xylan(Sigma Chemical Co., Inc., St. Louis, Mo., USA) by xylan-degradingenzyme(s) under the following typical conditions: 1 ml reactions, 5mg/ml substrate (total solids), 5 mg of xylanolytic protein/g ofsubstrate, 50 mM sodium acetate pH 5, 50° C., 24 hours, sugar analysisusing p-hydroxybenzoic acid hydrazide (PHBAH) assay as described byLever, 1972, A new reaction for colorimetric determination ofcarbohydrates, Anal. Biochem 47: 273-279.

Xylanase: The term “xylanase” means a 1,4-beta-D-xylan-xylohydrolase(E.C. 3.2.1.8) that catalyzes the endohydrolysis of 1,4-beta-D-xylosidiclinkages in xylans. For purposes of the present invention, xylanaseactivity is determined with 0.2% AZCL-arabinoxylan as substrate in 0.01%TRITON® X-100 and 200 mM sodium phosphate buffer pH 6 at 37° C. One unitof xylanase activity is defined as 1.0 μmole of azurine produced perminute at 37° C., pH 6 from 0.2% AZCL-arabinoxylan as substrate in 200mM sodium phosphate pH 6 buffer.

Beta-xylosidase: The term “beta-xylosidase” means a beta-D-xylosidexylohydrolase (E.C. 3.2.1.37) that catalyzes the exo-hydrolysis of shortbeta (1→4)-xylooligosaccharides, to remove successive D-xylose residuesfrom the non-reducing termini. For purposes of the present invention,one unit of beta-xylosidase is defined as 1.0 μmole of p-nitrophenolateanion produced per minute at 40° C., pH 5 from 1 mMp-nitrophenyl-beta-D-xyloside as substrate in 100 mM sodium citratecontaining 0.01% TWEEN® 20.

Acetylxylan esterase: The term “acetylxylan esterase” means acarboxylesterase (EC 3.1.1.72) that catalyzes the hydrolysis of acetylgroups from polymeric xylan, acetylated xylose, acetylated glucose,alpha-napthyl acetate, and p-nitrophenyl acetate. For purposes of thepresent invention, acetylxylan esterase activity is determined using 0.5mM p-nitrophenylacetate as substrate in 50 mM sodium acetate pH 5.0containing 0.01% TWEEN™ 20. One unit of acetylxylan esterase is definedas the amount of enzyme capable of releasing 1 μmole of p-nitrophenolateanion per minute at pH 5, 25° C.

Feruloyl esterase: The term “feruloyl esterase” means a4-hydroxy-3-methoxycinnamoyl-sugar hydrolase (EC 3.1.1.73) thatcatalyzes the hydrolysis of the 4-hydroxy-3-methoxycinnamoyl (feruloyl)group from an esterified sugar, which is usually arabinose in “natural”substrates, to produce ferulate (4-hydroxy-3-methoxycinnamate). Feruloylesterase is also known as ferulic acid esterase, hydroxycinnamoylesterase, FAE-III, cinnamoyl ester hydrolase, FAEA, cinnAE, FAE-I, orFAE-II. For purposes of the present invention, feruloyl esteraseactivity is determined using 0.5 mM p-nitrophenylferulate as substratein 50 mM sodium acetate pH 5.0. One unit of feruloyl esterase equals theamount of enzyme capable of releasing 1 μmole of p-nitrophenolate anionper minute at pH 5, 25° C.

Alpha-glucuronidase: The term “alpha-glucuronidase” means analpha-D-glucosiduronate glucuronohydrolase (EC 3.2.1.139) that catalyzesthe hydrolysis of an alpha-D-glucuronoside to D-glucuronate and analcohol. For purposes of the present invention, alpha-glucuronidaseactivity is determined according to de Vries, 1998, J. Bacteriol. 180:243-249. One unit of alpha-glucuronidase equals the amount of enzymecapable of releasing 1 μmole of glucuronic or 4-O-methylglucuronic acidper minute at pH 5, 40° C.

Alpha-L-arabinofuranosidase: The term “alpha-L-arabinofuranosidase”means an alpha-L-arabinofuranoside arabinofuranohydrolase (EC 3.2.1.55)that catalyzes the hydrolysis of terminal non-reducingalpha-L-arabinofuranoside residues in alpha-L-arabinosides. The enzymeacts on alpha-L-arabinofuranosides, alpha-L-arabinans containing (1,3)-and/or (1,5)-linkages, arabinoxylans, and arabinogalactans.Alpha-L-arabinofuranosidase is also known as arabinosidase,alpha-arabinosidase, alpha-L-arabinosidase, alpha-arabinofuranosidase,polysaccharide alpha-L-arabinofuranosidase, alpha-L-arabinofuranosidehydrolase, L-arabinosidase, or alpha-L-arabinanase. For purposes of thepresent invention, alpha-L-arabinofuranosidase activity is determinedusing 5 mg of medium viscosity wheat arabinoxylan (MegazymeInternational Ireland, Ltd., Bray, Co. Wicklow, Ireland) per ml of 100mM sodium acetate pH 5 in a total volume of 200 μl for 30 minutes at 40°C. followed by arabinose analysis by AMINEX® HPX-87H columnchromatography (Bio-Rad Laboratories, Inc., Hercules, Calif., USA).

Cellulosic material: The term “cellulosic material” means any materialcontaining cellulose. The predominant polysaccharide in the primary cellwall of biomass is cellulose, the second most abundant is hemicellulose,and the third is pectin. The secondary cell wall, produced after thecell has stopped growing, also contains polysaccharides and isstrengthened by polymeric lignin covalently cross-linked tohemicellulose. Cellulose is a homopolymer of anhydrocellobiose and thusa linear beta-(1-4)-D-glucan, while hemicelluloses include a variety ofcompounds, such as xylans, xyloglucans, arabinoxylans, and mannans incomplex branched structures with a spectrum of substituents. Althoughgenerally polymorphous, cellulose is found in plant tissue primarily asan insoluble crystalline matrix of parallel glucan chains.Hemicelluloses usually hydrogen bond to cellulose, as well as to otherhemicelluloses, which help stabilize the cell wall matrix.

Cellulose is generally found, for example, in the stems, leaves, hulls,husks, and cobs of plants or leaves, branches, and wood of trees. Thecellulosic material can be, but is not limited to, herbaceous material,agricultural residue, forestry residue, municipal solid waste, wastepaper, and pulp and paper mill residue (see, for example, Wiselogel etal., 1995, in Handbook on Bioethanol (Charles E. Wyman, editor), pp.105-118, Taylor & Francis, Washington D.C.; Wyman, 1994, BioresourceTechnology 50: 3-16; Lynd, 1990, Applied Biochemistry and Biotechnology24/25: 695-719; Mosier et al., 1999, Recent Progress in Bioconversion ofLignocellulosics, in Advances in Biochemical Engineering/Biotechnology,T. Scheper, managing editor, Volume 65, pp. 23-40, Springer-Verlag, NewYork). It is understood herein that the cellulose may be in the form oflignocellulose, a plant cell wall material containing lignin, cellulose,and hemicellulose in a mixed matrix. In a preferred aspect, thecellulosic material is lignocellulose, which comprises cellulose,hemicellulose, and lignin.

In one aspect, the cellulosic material is herbaceous material. Inanother aspect, the cellulosic material is agricultural residue. Inanother aspect, the cellulosic material is forestry residue. In anotheraspect, the cellulosic material is municipal solid waste. In anotheraspect, the cellulosic material is waste paper. In another aspect, thecellulosic material is pulp and paper mill residue.

In another aspect, the cellulosic material is corn stover. In anotheraspect, the cellulosic material is corn fiber. In another aspect, thecellulosic material is corn cob. In another aspect, the cellulosicmaterial is orange peel. In another aspect, the cellulosic material isrice straw. In another aspect, the cellulosic material is wheat straw.In another aspect, the cellulosic material is switch grass. In anotheraspect, the cellulosic material is miscanthus. In another aspect, thecellulosic material is bagasse.

In another aspect, the cellulosic material is microcrystallinecellulose. In another aspect, the cellulosic material is bacterialcellulose. In another aspect, the cellulosic material is algalcellulose. In another aspect, the cellulosic material is cotton linter.In another aspect, the cellulosic material is amorphous phosphoric-acidtreated cellulose. In another aspect, the cellulosic material is filterpaper.

The cellulosic material may be used as is or may be subjected topretreatment, using conventional methods known in the art, as describedherein. In a preferred aspect, the cellulosic material is pretreated.

Pretreated corn stover: The term “PCS” or “Pretreated Corn Stover” meansa cellulosic material derived from corn stover by treatment with heatand dilute sulfuric acid.

Xylan-containing material: The term “xylan-containing material” meansany material comprising a plant cell wall polysaccharide containing abackbone of beta-(1-4)-linked xylose residues. Xylans of terrestrialplants are heteropolymers possessing a beta-(1-4)-D-xylopyranosebackbone, which is branched by short carbohydrate chains. They compriseD-glucuronic acid or its 4-O-methyl ether, L-arabinose, and/or variousoligosaccharides, composed of D-xylose, L-arabinose, D- or L-galactose,and D-glucose. Xylan-type polysaccharides can be divided into homoxylansand heteroxylans, which include glucuronoxylans,(arabino)glucuronoxylans, (glucurono)arabinoxylans, arabinoxylans, andcomplex heteroxylans. See, for example, Ebringerova et al., 2005, Adv.Polym. Sci. 186: 1-67.

In the methods of the present invention, any material containing xylanmay be used. In a preferred aspect, the xylan-containing material islignocellulose.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. In one aspect, the maturepolypeptide is amino acids 18 to 481 of SEQ ID NO: 2 based on theSignalP program (Nielsen et al., 1997, Protein Engineering 10: 1-6) thatpredicts amino acids 1 to 17 of SEQ ID NO: 2 are a signal peptide. It isknown in the art that a host cell may produce a mixture of two of moredifferent mature polypeptides (i.e., with a different C-terminal and/orN-terminal amino acid) expressed by the same polynucleotide.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” means a polynucleotide that encodes a mature polypeptidehaving cellobiohydrolase II activity. In one aspect, the maturepolypeptide coding sequence is nucleotides 52 to 1443 of SEQ ID NO: 1based on the SignalP [program, e.g., (Nielsen et al., 1997, ProteinEngineering 10: 1-6) that predicts nucleotides 1 to 51 of SEQ ID NO: 1encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is the cDNA sequence contained in nucleotides 52 to 1443of SEQ ID NO: 1.

Sequence identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the degree of sequence identitybetween two amino acid sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol.48: 443-453) as implemented in the Needle program of the EMBOSS package(EMBOSS: The European Molecular Biology Open Software Suite, Rice etal., 2000, Trends Genet. 16: 276-277), preferably version 3.0.0 orlater. The optional parameters used are gap open penalty of 10, gapextension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62)substitution matrix. The output of Needle labeled “longest identity”(obtained using the −nobrief option) is used as the percent identity andis calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the degree of sequence identitybetween two deoxyribonucleotide sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,supra), preferably version 3.0.0 or later. The optional parameters usedare gap open penalty of 10, gap extension penalty of 0.5, and theEDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The outputof Needle labeled “longest identity” (obtained using the −nobriefoption) is used as the percent identity and is calculated as follows:

(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Fragment: The term “fragment” means a polypeptide having one or more(several) amino acids deleted from the amino and/or carboxyl terminus ofa mature polypeptide; wherein the fragment has cellobiohydrolase IIactivity. In one aspect, a fragment contains at least 390 amino acidresidues, e.g., at least 415 amino acid residues or at least 440 aminoacid residues.

Subsequence: The term “subsequence” means a polynucleotide having one ormore (several) nucleotides deleted from the 5′- and/or 3′-end of amature polypeptide coding sequence; wherein the subsequence encodes afragment having cellobiohydrolase II activity. In one aspect, asubsequence contains at least 1170 nucleotides, e.g., at least 1245nucleotides or at least 1320 nucleotides.

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of its polypeptideproduct. The boundaries of the coding sequence are generally determinedby an open reading frame, which usually begins with the ATG start codonor alternative start codons such as GTG and TTG and ends with a stopcodon such as TAA, TAG, and TGA. The coding sequence may be a DNA, cDNA,synthetic, or recombinant polynucleotide.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic cell. cDNA lacks intron sequences that may be presentin the corresponding genomic DNA. The initial, primary RNA transcript isa precursor to mRNA that is processed through a series of steps,including splicing, before appearing as mature spliced mRNA.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic. The term nucleic acid construct issynonymous with the term “expression cassette” when the nucleic acidconstruct contains the control sequences required for expression of acoding sequence of the present invention.

Control sequences: The term “control sequences” means all componentsnecessary for the expression of a polynucleotide encoding a variant ofthe present invention. Each control sequence may be native or foreign tothe polynucleotide encoding the variant or native or foreign to eachother. Such control sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter, signal peptidesequence, and transcription terminator. At a minimum, the controlsequences include a promoter, and transcriptional and translational stopsignals. The control sequences may be provided with linkers for thepurpose of introducing specific restriction sites facilitating ligationof the control sequences with the coding region of the polynucleotideencoding a variant.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs the expression of the coding sequence.

Expression: The term “expression” includes any step involved in theproduction of a variant of the present invention including, but notlimited to, transcription, post-transcriptional modification,translation, post-translational modification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding a variantof the present invention and is operably linked to additionalnucleotides that provide for its expression.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, and the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Increased thermostability: The term “increased thermostability” means ahigher retention of cellobiohydrolase II activity of a variant after aperiod of incubation at a temperature relative to the parent. Theincreased thermostability of the variant relative to the parent can beassessed, for example, under conditions of one or more (several)temperatures. For example, the one or more (several) temperatures can beany temperature in the range of 45° C. to 95° C., e.g., 45, 50, 55, 60,65, 70, 75, 80, 85, or 95° C. (or in between, e.g., 67° C.) at a pH inthe range of 3 to 8, e.g., 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0,7.5, or 8.0, (or in between) for a suitable period of incubation, e.g.,1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, or60 minutes, such that the variant retains residual activity relative tothe parent.

In one aspect, the thermostability of the variant relative to the parentis determined at pH 3.0 and 50° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 3.0 and 55° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 3.0 and 60° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 3.0 and 65° C. In another aspect, the thermostabilityof the variant relative to the parent is determined at pH 3.0 and 70° C.In another aspect, the thermostability of the variant relative to theparent is determined at pH 3.0 and 75° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 3.0 and 80° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 3.0 and 85° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 3.0 and 90° C.

In another aspect, the thermostability of the variant relative to theparent is determined at pH 3.5 and 50° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 3.5 and 55° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 3.5 and 60° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 3.5 and 65° C. In another aspect, the thermostabilityof the variant relative to the parent is determined at pH 3.5 and 70° C.In another aspect, the thermostability of the variant relative to theparent is determined at pH 3.5 and 75° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 3.5 and 80° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 3.5 and 85° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 3.5 and 90° C.

In another aspect, the thermostability of the variant relative to theparent is determined at pH 4.0 and 50° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 4.0 and 55° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 4.0 and 60° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 4.0 and 65° C. In another aspect, the thermostabilityof the variant relative to the parent is determined at pH 4.0 and 70° C.In another aspect, the thermostability of the variant relative to theparent is determined at pH 4.0 and 75° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 4.0 and 80° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 4.0 and 85° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 4.0 and 90° C.

In another aspect, the thermostability of the variant relative to theparent is determined at pH 4.5 and 50° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 4.5 and 55° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 4.5 and 60° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 4.5 and 65° C. In another aspect, the thermostabilityof the variant relative to the parent is determined at pH 4.5 and 70° C.In another aspect, the thermostability of the variant relative to theparent is determined at pH 4.5 and 75° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 4.5 and 80° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 4.5 and 85° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 4.5 and 90° C.

In another aspect, the thermostability of the variant relative to theparent is determined at pH 5.0 and 50° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 5.0 and 55° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 5.0 and 60° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 5.0 and 65° C. In another aspect, the thermostabilityof the variant relative to the parent is determined at pH 5.0 and 70° C.In another aspect, the thermostability of the variant relative to theparent is determined at pH 5.0 and 75° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 5.0 and 80° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 5.0 and 85° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 5.0 and 90° C.

In another aspect, the thermostability of the variant relative to theparent is determined at pH 5.5 and 50° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 5.5 and 55° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 5.5 and 60° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 5.5 and 65° C. In another aspect, the thermostabilityof the variant relative to the parent is determined at pH 5.5 and 70° C.In another aspect, the thermostability of the variant relative to theparent is determined at pH 5.5 and 75° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 5.5 and 80° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 5.5 and 85° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 5.5 and 90° C.

In another aspect, the thermostability of the variant relative to theparent is determined at pH 6.0 and 50° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 6.0 and 55° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 6.0 and 60° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 6.0 and 65° C. In another aspect, the thermostabilityof the variant relative to the parent is determined at pH 6.0 and 70° C.In another aspect, the thermostability of the variant relative to theparent is determined at pH 6.0 and 75° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 6.0 and 80° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 6.0 and 85° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 6.0 and 90° C.

In another aspect, the thermostability of the variant relative to theparent is determined at pH 6.5 and 50° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 6.5 and 55° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 6.5 and 60° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 6.5 and 65° C. In another aspect, the thermostabilityof the variant relative to the parent is determined at pH 6.5 and 70° C.In another aspect, the thermostability of the variant relative to theparent is determined at pH 6.5 and 75° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 6.5 and 80° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 6.5 and 85° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 6.5 and 90° C.

In another aspect, the thermostability of the variant relative to theparent is determined at pH 7.0 and 50° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 7.0 and 55° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 7.0 and 60° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 7.0 and 65° C. In another aspect, the thermostabilityof the variant relative to the parent is determined at pH 7.0 and 70° C.In another aspect, the thermostability of the variant relative to theparent is determined at pH 7.0 and 75° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 7.0 and 80° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 7.0 and 85° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 7.0 and 90° C.

In another aspect, the thermostability of the variant relative to theparent is determined at pH 7.5 and 50° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 7.5 and 55° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 7.5 and 60° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 7.5 and 65° C. In another aspect, the thermostabilityof the variant relative to the parent is determined at pH 7.5 and 70° C.In another aspect, the thermostability of the variant relative to theparent is determined at pH 7.5 and 75° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 7.5 and 80° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 7.5 and 85° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 7.5 and 90° C.

In another aspect, the thermostability of the variant relative to theparent is determined at pH 8.0 and 50° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 8.0 and 55° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 8.0 and 60° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 8.0 and 65° C. In another aspect, the thermostabilityof the variant relative to the parent is determined at pH 8.0 and 70° C.In another aspect, the thermostability of the variant relative to theparent is determined at pH 8.0 and 75° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 8.0 and 80° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 8.0 and 85° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 8.0 and 90° C.

In each of the aspects above, the thermostability of the variantrelative to the parent is determined by incubating the variant andparent for 1 minute. In each of the aspects above, the thermostabilityof the variant relative to the parent is determined by incubating thevariant and parent for 5 minutes. In each of the aspects above, thethermostability of the variant relative to the parent is determined byincubating the variant and parent for 10 minutes. In each of the aspectsabove, the thermostability of the variant relative to the parent isdetermined by incubating the variant and parent for 15 minutes. In eachof the aspects above, the thermostability of the variant relative to theparent is determined by incubating the variant and parent for 30minutes. In each of the aspects above, the thermostability of thevariant relative to the parent is determined by incubating the variantand parent for 45 minutes. In each of the aspects above, thethermostability of the variant relative to the parent is determined byincubating the variant and parent for 60 minutes. However, any timeperiod can be used to demonstrate increased thermostability of a variantof the present invention relative to the parent.

The increased thermostability of the variant relative to the parent canbe determined by differential scanning calorimetry (DSC) using methodsstandard in the art (see, for example, Sturtevant, 1987, Annual Reviewof Physical Chemistry 38: 463-488). The increased thermostability of thevariant relative to the parent can also be determined using any enzymeassay known in the art for cellobiohydrolase II. The increasedthermostability of the variant relative to the parent can also bedetermined using the assay described in Example 5.

In one aspect, the thermostability of the variant havingcellobiohydrolase II activity is at least 1.05-fold, e.g., at least1.1-fold, at least 1.5-fold, at least 1.8-fold, at least 2-fold, atleast 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, atleast 25-fold, and at least 50-fold more thermostable than the parent.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to isolated variants of a parentcellobiohydrolase II, comprising a substitution at one or more (several)positions corresponding to positions 272, 287, 325, 347, 357, 363, 409,464, and 476 of the mature polypeptide of SEQ ID NO: 2, wherein thevariant has cellobiohydrolase II activity. A variant of the presentinvention has increased thermostability compared to the parent.

Conventions for Designation of Variants

For purposes of the present invention, the mature polypeptide disclosedin SEQ ID NO: 2 is used to determine the corresponding amino acidresidue in another cellobiohydrolase II. The amino acid sequence ofanother cellobiohydrolase II is aligned with the mature polypeptidedisclosed in SEQ ID NO: 2, and based on the alignment, the amino acidposition number corresponding to any amino acid residue in the maturepolypeptide disclosed in SEQ ID NO: 2 is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol.48: 443-453) as implemented in the Needle program of the EMBOSS package(EMBOSS: The European Molecular Biology Open Software Suite, Rice etal., 2000, Trends Genet. 16: 276-277), preferably version 3.0.0 orlater.

Identification of the corresponding amino acid residue in anothercellobiohydrolase II can be confirmed by an alignment of multiplepolypeptide sequences using “ClustalW” (Larkin et al., 2007,Bioinformatics 23: 2947-2948).

When the other enzyme has diverged from the mature polypeptide of SEQ IDNO: 2 such that traditional sequence-based comparison fails to detecttheir relationship (Lindahl and Elofsson, 2000, J. Mol. Biol. 295:613-615), other pairwise sequence comparison algorithms can be used.Greater sensitivity in sequence-based searching can be attained usingsearch programs that utilize probabilistic representations ofpolypeptide families (profiles) to search databases. For example, thePSI-BLAST program generates profiles through an iterative databasesearch process and is capable of detecting remote homologs (Atschul etal., 1997, Nucleic Acids Res. 25: 3389-3402). Even greater sensitivitycan be achieved if the family or superfamily for the polypeptide has oneor more representatives in the protein structure databases. Programssuch as GenTHREADER (Jones, 1999, J. Mol. Biol. 287: 797-815; McGuffinand Jones, 2003, Bioinformatics 19: 874-881) utilize information from avariety of sources (PSI-BLAST, secondary structure prediction,structural alignment profiles, and solvation potentials) as input to aneural network that predicts the structural fold for a query sequence.Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313: 903-919,can be used to align a sequence of unknown structure with thesuperfamily models present in the SCOP database. These alignments can inturn be used to generate homology models for the polypeptide, and suchmodels can be assessed for accuracy using a variety of tools developedfor that purpose.

For proteins of known structure, several tools and resources areavailable for retrieving and generating structural alignments. Forexample the SCOP superfamilies of proteins have been structurallyaligned, and those alignments are accessible and downloadable. Two ormore protein structures can be aligned using a variety of algorithmssuch as the distance alignment matrix (Holm and Sander, 1998, Proteins33: 88-96) or combinatorial extension (Shindyalov and Bourne, 1998,Protein Engineering 11: 739-747), and implementation of these algorithmscan additionally be utilized to query structure databases with astructure of interest in order to discover possible structural homologs(e.g., Holm and Park, 2000, Bioinformatics 16: 566-567).

In describing the variants of the present invention, the nomenclaturedescribed below is adapted for ease of reference. The accepted IUPACsingle letter or three letter amino acid abbreviation is employed.

Substitutions.

For an amino acid substitution, the following nomenclature is used:Original amino acid, position, substituted amino acid. Accordingly, thesubstitution of threonine with alanine at position 226 is designated as“Thr226Ala” or “T226A”. Multiple mutations are separated by additionmarks (“+”), e.g., “Gly205Arg+Ser411Phe” or “G205R+S411F”, representingsubstitutions at positions 205 and 411 of glycine (G) with arginine (R)and serine (S) with phenylalanine (F), respectively.

Deletions.

For an amino acid deletion, the following nomenclature is used: Originalamino acid, position*. Accordingly, the deletion of glycine at position195 is designated as “Gly195*” or “G195*”. Multiple deletions areseparated by addition marks (“+”), e.g., “Gly195*+Ser411*” or“G195*+S411*”.

Insertions.

For an amino acid insertion, the following nomenclature is used:Original amino acid, position, original amino acid, inserted amino acid.Accordingly the insertion of lysine after glycine at position 195 isdesignated “Gly195GlyLys” or “G195GK”. An insertion of multiple aminoacids is designated [Original amino acid, position, original amino acid,inserted amino acid #1, inserted amino acid #2; etc.]. For example, theinsertion of lysine and alanine after glycine at position 195 isindicated as “Gly195GlyLysAla” or “G195GKA”.

In such cases the inserted amino acid residue(s) are numbered by theaddition of lower case letters to the position number of the amino acidresidue preceding the inserted amino acid residue(s). In the aboveexample, the sequence would thus be:

Parent: Variant: 195 195 195a 195b G G-K-A

Multiple Alterations.

Variants comprising multiple alterations are separated by addition marks(“+”), e.g., “Arg170Tyr+Gly195Glu” or “R170Y+G195E” representing asubstitution of tyrosine and glutamic acid for arginine and glycine atpositions 170 and 195, respectively.

Different Alterations.

Where different alterations can be introduced at a position, thedifferent alterations are separated by a comma, e.g., “Arg170Tyr,Glu”represents a substitution of arginine with tyrosine or glutamic acid atposition 170. Thus, “Tyr167Gly,Ala+Arg170Gly,Ala” designates thefollowing variants:

“Tyr167Gly+Arg170Gly”, “Tyr167Gly+Arg170Ala”, “Tyr167Ala+Arg170Gly”, and“Tyr167Ala+Arg170Ala”.

Cellobiohydrolase II Parents

The parent cellobiohydrolase II may be (a) a polypeptide having at least60% sequence identity to the mature polypeptide of SEQ ID NO: 2; (b) apolypeptide encoded by a polynucleotide that hybridizes under lowstringency conditions with (i) the mature polypeptide coding sequence ofSEQ ID NO: 1, (ii) the genomic DNA sequence of the mature polypeptidecoding sequence of SEQ ID NO: 1, or (iii) the full-length complementarystrand of (i) or (ii); or (c) a polypeptide encoded by a polynucleotidehaving at least 60% sequence identity to the mature polypeptide codingsequence of SEQ ID NO: 1 or the genomic DNA sequence thereof.

In a first aspect, the parent has a sequence identity to the maturepolypeptide of SEQ ID NO: 2 of at least 60%, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%, which havecellobiohydrolase II activity. In one aspect, the amino acid sequence ofthe parent differs by no more than ten amino acids, e.g., by five aminoacids, by four amino acids, by three amino acids, by two amino acids,and by one amino acid from the mature polypeptide of SEQ ID NO: 2.

In one aspect, the parent comprises or consists of the amino acidsequence of SEQ ID NO: 2. In another aspect, the parent comprises orconsists of the mature polypeptide of SEQ ID NO: 2. In another aspect,the parent comprises or consists of amino acids 18 to 481 of SEQ ID NO:2.

In an embodiment, the parent is a fragment of the mature polypeptide ofSEQ ID NO: 2 containing at least 390 amino acid residues, e.g., at least415 amino acid residues or at least 440 amino acid residues.

In another embodiment, the parent is an allelic variant of the maturepolypeptide of SEQ ID NO: 2.

In a second aspect, the parent is encoded by a polynucleotide thathybridizes under very low stringency conditions, low stringencyconditions, medium stringency conditions, medium-high stringencyconditions, high stringency conditions, or very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, (ii) the genomic DNA sequence of the mature polypeptide codingsequence of SEQ ID NO: 1, or (iii) the full-length complementary strandof (i) or (ii) (J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989,Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor,New York).

The polynucleotide of SEQ ID NO: 1 or a subsequence thereof, as well asthe amino acid sequence of SEQ ID NO: 2 or a fragment thereof, may beused to design nucleic acid probes to identify and clone DNA encoding aparent from strains of different genera or species according to methodswell known in the art. In particular, such probes can be used forhybridization with the genomic or cDNA of the genus or species ofinterest, following standard Southern blotting procedures, in order toidentify and isolate the corresponding gene therein. Such probes can beconsiderably shorter than the entire sequence, but should be at least14, e.g., at least 25, at least 35, or at least 70 nucleotides inlength. Preferably, the nucleic acid probe is at least 100 nucleotidesin length, e.g., at least 200 nucleotides, at least 300 nucleotides, atleast 400 nucleotides, at least 500 nucleotides, at least 600nucleotides, at least 700 nucleotides, at least 800 nucleotides, or atleast 900 nucleotides in length. Both DNA and RNA probes can be used.The probes are typically labeled for detecting the corresponding gene(for example, with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes areencompassed by the present invention.

A genomic DNA or cDNA library prepared from such other organisms may bescreened for DNA that hybridizes with the probes described above andencodes a parent. Genomic or other DNA from such other organisms may beseparated by agarose or polyacrylamide gel electrophoresis, or otherseparation techniques. DNA from the libraries or the separated DNA maybe transferred to and immobilized on nitrocellulose or other suitablecarrier material. In order to identify a clone or DNA that hybridizeswith SEQ ID NO: 1 or a subsequence thereof, the carrier material is usedin a Southern blot.

For purposes of the present invention, hybridization indicates that thepolynucleotide hybridizes to a labeled nucleotide probe corresponding tothe polynucleotide shown in SEQ ID NO: 1 or the genomic DNA sequencethereof, its full-length complementary strand, or a subsequence thereof,under low to very high stringency conditions. Molecules to which theprobe hybridizes can be detected using, for example, X-ray film or anyother detection means known in the art.

In one aspect, the nucleic acid probe is the mature polypeptide codingsequence of SEQ ID NO: 1 or the genomic DNA sequence thereof. In anotheraspect, the nucleic acid probe is nucleotides 52 to 1443 of SEQ ID NO: 1or the genomic DNA sequence thereof. In another aspect, the nucleic acidprobe is a polynucleotide that encodes the polypeptide of SEQ ID NO: 2or the mature polypeptide thereof, or a fragment thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 1 or the genomic DNAsequence thereof.

For long probes of at least 100 nucleotides in length, very low to veryhigh stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and either 25% formamide for very lowand low stringencies, 35% formamide for medium and medium-highstringencies, or 50% formamide for high and very high stringencies,following standard Southern blotting procedures for 12 to 24 hoursoptimally. The carrier material is finally washed three times each for15 minutes using 2×SSC, 0.2% SDS at 45° C. (very low stringency), 50° C.(low stringency), 55° C. (medium stringency), 60° C. (medium-highstringency), 65° C. (high stringency), or 70° C. (very high stringency).

For short probes that are about 15 nucleotides to about 70 nucleotidesin length, stringency conditions are defined as prehybridization andhybridization at about 5° C. to about 10° C. below the calculated T_(m)using the calculation according to Bolton and McCarthy (1962, Proc.Natl. Acad. Sci. USA 48: 1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6mM EDTA, 0.5% NP-40, 1×Denhardt's solution, 1 mM sodium pyrophosphate, 1mM sodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA perml following standard Southern blotting procedures for 12 to 24 hoursoptimally. The carrier material is finally washed once in 6×SCC plus0.1% SDS for 15 minutes and twice each for 15 minutes using 6×SSC at 5°C. to 10° C. below the calculated T_(m).

In a third aspect, the parent is encoded by a polynucleotide having asequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1 or the genomic DNA sequence thereof of at least 60%, e.g., atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100%, which encodes a polypeptide having cellobiohydrolase II activity.In one aspect, the mature polypeptide coding sequence is nucleotides 52to 1443 of SEQ ID NO: 1 or the genomic DNA sequence thereof. In anembodiment, the parent is encoded by a polynucleotide comprising orconsisting of SEQ ID NO: 1 or the genomic DNA sequence thereof.

The parent may be obtained from microorganisms of any genus. Forpurposes of the present invention, the term “obtained from” as usedherein in connection with a given source shall mean that the parentencoded by a polynucleotide is produced by the source or by a cell inwhich the polynucleotide from the source has been inserted. In oneaspect, the parent is secreted extracellularly.

The parent may be a bacterial cellobiohydrolase II. For example, theparent may be a gram-positive bacterial polypeptide such as a Bacillus,Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus,Oceanobacillus, Staphylococcus, Streptococcus, or Streptomycescellobiohydrolase II, or a gram-negative bacterial polypeptide such as aCampylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, or Ureaplasmacellobiohydrolase II.

In one aspect, the parent is a Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillusclausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacilluslentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus,Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis cellobiohydrolase II.

In another aspect, the parent is a Streptococcus equisimilis,Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equisubsp. Zooepidemicus cellobiohydrolase II.

In another aspect, the parent is a Streptomyces achromogenes,Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus,or Streptomyces lividans cellobiohydrolase II. The parent may be afungal cellobiohydrolase II. For example, the parent may be a yeastcellobiohydrolase II such as a Candida, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cellobiohydrolase II.For example, the parent may be a filamentous fungal cellobiohydrolase IIsuch as an Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium,Botryosphaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps,Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria,Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella,Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria,Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora,Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete,Piromyces, Poitrasia, Pseudoplectania, Pseudotrichonympha, Rhizomucor,Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia,Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella, orXylaria cellobiohydrolase II.

In another aspect, the parent is a Saccharomyces carlsbergensis,Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomycesdouglasii, Saccharomyces kluyveri, Saccharomyces norbensis, orSaccharomyces oviformis cellobiohydrolase II.

In another aspect, the parent is an Acremonium cellulolyticus,Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus,Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans,Aspergillus niger, Aspergillus oryzae, Chrysosporium inops,Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporiummerdarium, Chrysosporium pannicola, Chrysosporium queenslandicum,Chrysosporium tropicum, Chrysosporium zonatum, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa,Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurosporacrassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaetechrysosporium, Thielavia achromatica, Thielavia albomyces, Thielaviaalbopilosa, Thielavia australeinsis, Thielavia fimeti, Thielaviamicrospora, Thielavia ovispora, Thielavia peruviana, Thielavia setosa,Thielavia spededonium, Thielavia subthermophila, Thielavia terrestris,Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, or Trichoderma viridecellobiohydrolase II.

In another aspect, the parent is a Thielavia cellobiohydrolase II. Inanother aspect, the parent is a Thielavia terrestris cellobiohydrolaseII. In another aspect, the parent is the Thielavia terrestriscellobiohydrolase II of SEQ ID NO: 2 or the mature polypeptide thereof.In another aspect, the parent cellobiohydrolase II is encoded by thenucleotide sequence contained in plasmid pTter6A which is contained inE. coli NRRL B-30802. In another aspect, the parent cellobiohydrolase IIis encoded by the mature polypeptide coding sequence contained inplasmid pTter6A which is contained in E. coli NRRL B-30802.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), andAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The parent may be identified and obtained from other sources includingmicroorganisms isolated from nature (e.g., soil, composts, water, etc.)or DNA samples obtained directly from natural materials (e.g., soil,composts, water, etc,) using the above-mentioned probes. Techniques forisolating microorganisms and DNA directly from natural habitats are wellknown in the art. The polynucleotide encoding a parent may then bederived by similarly screening a genomic or cDNA library of anothermicroorganism or mixed DNA sample. Once a polynucleotide encoding aparent has been detected with a probe(s), the polynucleotide may beisolated or cloned by utilizing techniques that are known to those ofordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).

The parent may be a hybrid polypeptide in which a portion of onepolypeptide is fused at the N-terminus or the C-terminus of a portion ofanother polypeptide.

The parent also may be a fused polypeptide or cleavable fusionpolypeptide in which one polypeptide is fused at the N-terminus or theC-terminus of another polypeptide. A fused polypeptide is produced byfusing a polynucleotide encoding one polypeptide to a polynucleotideencoding another polypeptide. Techniques for producing fusionpolypeptides are known in the art, and include ligating the codingsequences encoding the polypeptides so that they are in frame and thatexpression of the fused polypeptide is under control of the samepromoter(s) and terminator. Fusion proteins may also be constructedusing intein technology in which fusions are createdpost-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawsonet al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between thetwo polypeptides. Upon secretion of the fusion protein, the site iscleaved releasing the two polypeptides. Examples of cleavage sitesinclude, but are not limited to, the sites disclosed in Martin et al.,2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000,J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl.Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton etal., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995,Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure,Function, and Genetics 6: 240-248; and Stevens, 2003, Drug DiscoveryWorld 4: 35-48.

Preparation of Variants

The present invention also relates to methods for obtaining a varianthaving cellobiohydrolase II activity, comprising: (a) introducing into aparent cellobiohydrolase II a substitution at one or more (several)positions corresponding to positions 272, 287, 325, 347, 357, 363, 409,464, and 476 of the mature polypeptide of SEQ ID NO: 2, wherein thevariant has cellobiohydrolase II activity; and (b) recovering thevariant. In one aspect, a substitution is further introduced at aposition corresponding to position 435 of the mature polypeptide of SEQID NO: 2.

The variants can be prepared using any mutagenesis procedure known inthe art, such as site-directed mutagenesis, synthetic gene construction,semi-synthetic gene construction, random mutagenesis, shuffling, etc.

Site-directed mutagenesis is a technique in which one or more (several)mutations are created at one or more defined sites in a polynucleotideencoding the parent.

Site-directed mutagenesis can be accomplished in vitro by PCR involvingthe use of oligonucleotide primers containing the desired mutation.Site-directed mutagenesis can also be performed in vitro by cassettemutagenesis involving the cleavage by a restriction enzyme at a site inthe plasmid comprising a polynucleotide encoding the parent andsubsequent ligation of an oligonucleotide containing the mutation in thepolynucleotide. Usually the restriction enzyme that digests the plasmidand the oligonucleotide is the same, permitting sticky ends of theplasmid and insert to ligate to one another. See, e.g., Scherer andDavis, 1979, Proc. Natl. Acad. Sci. USA 76: 4949-4955; and Barton etal., 1990, Nucleic Acids Res. 18: 7349-4966.

Site-directed mutagenesis can also be accomplished in vivo by methodsknown in the art. See, e.g., U.S. Patent Application Publication No.2004/0171154; Storici et al., 2001, Nature Biotechnol. 19: 773-776; Krenet al., 1998, Nat. Med. 4: 285-290; and Calissano and Macino, 1996,Fungal Genet. Newslett. 43: 15-16.

Any site-directed mutagenesis procedure can be used in the presentinvention. There are many commercial kits available that can be used toprepare variants.

Synthetic gene construction entails in vitro synthesis of a designedpolynucleotide molecule to encode a polypeptide of interest. Genesynthesis can be performed utilizing a number of techniques, such as themultiplex microchip-based technology described by Tian et al. (2004,Nature 432: 1050-1054) and similar technologies wherein oligonucleotidesare synthesized and assembled upon photo-programmable microfluidicchips.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204) andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

Semi-synthetic gene construction is accomplished by combining aspects ofsynthetic gene construction, and/or site-directed mutagenesis, and/orrandom mutagenesis, and/or shuffling. Semi-synthetic construction istypified by a process utilizing polynucleotide fragments that aresynthesized, in combination with PCR techniques. Defined regions ofgenes may thus be synthesized de novo, while other regions may beamplified using site-specific mutagenic primers, while yet other regionsmay be subjected to error-prone PCR or non-error prone PCRamplification. Polynucleotide subsequences may then be shuffled.

Variants

The present invention also provides variants of a parentcellobiohydrolase II comprising a substitution at one or more (several)positions corresponding to positions 272, 287, 325, 347, 357, 363, 409,464, and 476, wherein the variant has cellobiohydrolase II activity.

In an embodiment, the variant has sequence identity of at least 60%,e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99%, but less than 100%, to the amino acid sequence of the parentcellobiohydrolase II.

In another embodiment, the variant has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, suchas at least 96%, at least 97%, at least 98%, or at least 99%, but lessthan 100%, sequence identity to the mature polypeptide of SEQ ID NO: 2.

In one aspect, the number of substitutions in the variants of thepresent invention is 1-9, e.g., such as 1, 2, 3, 4, 5, 6, 7, 8, or 9substitutions.

In one aspect, a variant comprises a substitution at one or more(several) positions corresponding to positions 272, 287, 325, 347, 357,363, 409, 464, and 476. In another aspect, a variant comprises asubstitution at two positions corresponding to any of positions 272,287, 325, 347, 357, 363, 409, 464, and 476. In another aspect, a variantcomprises a substitution at three positions corresponding to any ofpositions 272, 287, 325, 347, 357, 363, 409, 464, and 476. In anotheraspect, a variant comprises a substitution at four positionscorresponding to any of positions 272, 287, 325, 347, 357, 363, 409,464, and 476. In another aspect, a variant comprises a substitution atfive positions corresponding to any of positions 272, 287, 325, 347,357, 363, 409, 464, and 476. In another aspect, a variant comprises asubstitution at six positions corresponding to any of positions 272,287, 325, 347, 357, 363, 409, 464, and 476. In another aspect, a variantcomprises a substitution at seven positions corresponding to any ofpositions 272, 287, 325, 347, 357, 363, 409, 464, and 476. In anotheraspect, a variant comprises a substitution at eight positionscorresponding to any of positions 272, 287, 325, 347, 357, 363, 409,464, and 476. In another aspect, a variant comprises a substitution ateach position corresponding to positions 272, 287, 325, 347, 357, 363,409, 464, and 476.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 272. In another aspect, theamino acid at a position corresponding to position 272 is substitutedwith Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Ser. In anotheraspect, the variant comprises or consists of the substitution A272S ofthe mature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 287. In another aspect, theamino acid at a position corresponding to position 287 is substitutedwith Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Lys. In anotheraspect, the variant comprises or consists of the substitution Q287K ofthe mature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 325. In another aspect, theamino acid at a position corresponding to position 325 is substitutedwith Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Asp. In anotheraspect, the variant comprises or consists of the substitution S325D ofthe mature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 347. In another aspect, theamino acid at a position corresponding to position 347 is substitutedwith Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Ile. In anotheraspect, the variant comprises or consists of the substitution L347I ofthe mature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 357. In another aspect, theamino acid at a position corresponding to position 357 is substitutedwith Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met,Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Asn. In anotheraspect, the variant comprises or consists of the substitution D357N ofthe mature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 363. In another aspect, theamino acid at a position corresponding to position 363 is substitutedwith Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Lys. In anotheraspect, the variant comprises or consists of the substitution S363K ofthe mature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 409. In another aspect, theamino acid at a position corresponding to position 409 is substitutedwith Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Cys. In anotheraspect, the variant comprises or consists of the substitution G409C ofthe mature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 464. In another aspect, theamino acid at a position corresponding to position 464 is substitutedwith Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Gln. In anotheraspect, the variant comprises or consists of the substitution T464Q ofthe mature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 476. In another aspect, theamino acid at a position corresponding to position 476 is substitutedwith Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Cys. In anotheraspect, the variant comprises or consists of the substitution N476C ofthe mature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of a combination oftwo substitutions at positions corresponding to any of positions 272,287, 325, 347, 357, 363, 464, 409, and 476 of the mature polypeptide ofSEQ ID NO: 2. In another aspect, the variant comprises or consists of acombination of two substitutions at positions corresponding to any ofpositions 272, 287, 325, 347, 357, 363, 464, 409, and 476 of the maturepolypeptide of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. Inanother aspect, the variant comprises or consists of a combination oftwo substitutions of any of Ser, Lys, Asp, Ile, Asn, Lys, Gln, Cys, andCys at positions corresponding to positions 272, 287, 325, 347, 357,363, 464, 409, and 476, respectively, of the mature polypeptide of SEQID NO: 2. In another aspect, the variant comprises or consists of acombination of two substitutions of any of A272S, Q287K, S325D, L347I,D357N, S363K, G409C, T464Q, and N476C of the mature polypeptide of SEQID NO: 2.

The two positions are positions 272 and 287; 272 and 325; 272 and 347;272 and 357; 272 and 363; 272 and 409; 272 and 464; 272 and 476; 287 and325; 287 and 347; 287 and 357; 287 and 363; 287 and 409; 287 and 464;287 and 476; 325 and 347; 325 and 357; 325 and 363; 325 and 409; 325 and464; 325 and 476; 347 and 357; 347 and 363; 347 and 409; 347 and 464;347 and 476; 357 and 363; 357 and 409; 357 and 464; 357 and 476; 363 and409; 363 and 464; 363 and 476; 409 and 464; 409 and 476; or 464 and 476.

The combination of two substitutions is A272S and Q287K; A272S andS325D; A272S and L347I; A272S and D357N; A272S and S363K; A272S andG409C; A272S and T464Q; A272S and N476C; Q287K and S325D; Q287K andL347I; Q287K and D357N; Q287K and S363K; Q287K and G409C; Q287K andT464Q; Q287K and N476C; S325D and L347I; S325D and D357N; S325D andS363K; S325D and G409C; S325D and T464Q; S325D and N476C; L347I andD357N; L347I and S363K; L347I and G409C; L347I and T464Q; L347I andN476C; D357N and S363K; D357N and G409C; D357N and T464Q; D357N andN476C; S363K and G409C; S363K and T464Q; S363K and N476C; G409C andT464Q; G409C and N476C; or T464Q and N476C.

In another aspect, the variant comprises or consists of a combination ofthree substitutions at positions corresponding to any of positions 272,287, 325, 347, 357, 363, 464, 409, and 476 of the mature polypeptide ofSEQ ID NO: 2. In another aspect, the variant comprises or consists of acombination of three substitutions at positions corresponding to any ofpositions 272, 287, 325, 347, 357, 363, 464, 409, and 476 of the maturepolypeptide of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. Inanother aspect, the variant comprises or consists of a combination ofthree substitutions of any of Ser, Lys, Asp, Ile, Asn, Lys, Gin, Cys,and Cys at positions corresponding to positions 272, 287, 325, 347, 357,363, 464, 409, and 476, respectively, of the mature polypeptide of SEQID NO: 2. In another aspect, the variant comprises or consists of acombination of three substitutions of any of A272S, Q287K, S325D, L347I,D357N, S363K, T464Q, G409C, and N476C of the mature polypeptide of SEQID NO: 2.

The combination of three positions is positions 272, 287, and 325; 272,287, and 347; 272, 287, and 357; 272, 287, and 363; 272, 287, and 409;272, 287, and 464; 272, 287, and 476; 272, 325, and 347; 272, 325, and357; 272, 325, and 363; 272, 325, and 409; 272, 325, and 464; 272, 325,and 476; 272, 347, and 357; 272, 347, and 363; 272, 347, and 409; 272,347, and 464; 272, 347, and 476; 272, 357, and 363; 272, 357, and 409;272, 357, and 464; 272, 357, and 476; 272, 363, and 409; 272, 363, and464; 272, 363, and 476; 272, 409, and 464; 272, 409, and 476; 272, 464,and 476; 287, 325, and 347; 287, 325, and 357; 287, 325, and 363; 287,325, and 409; 287, 325, and 464; 287, 325, and 476; 287, 347, and 357;287, 347, and 363; 287, 347, and 409; 287, 347, and 464; 287, 347, and476; 287, 357, and 363; 287, 357, and 409; 287, 357, and 464; 287, 357,and 476; 287, 363, and 409; 287, 363, and 464; 287, 363, and 476; 287,409, and 464; 287, 409, and 476; 287, 464, and 476; 325, 347, and 357;325, 347, and 363; 325, 347, and 409; 325, 347, and 464; 325, 347, and476; 325, 357, and 363; 325, 357, and 409; 325, 357, and 464; 325, 357,and 476; 325, 363, and 409; 325, 363, and 464; 325, 363, and 476; 325,409, and 464; 325, 409, and 476; 325, 464, and 476; 347, 357, and 363;347, 357, and 409; 347, 357, and 464; 347, 357, and 476; 347, 363, and409; 347, 363, and 464; 347, 363, and 476; 347, 409, and 464; 347, 409,and 476; 347, 464, and 476; 357, 363, and 409; 357, 363, and 464; 357,363, and 476; 357, 409, and 464; 357, 409, and 476; 357, 464, and 476;363, 409, and 464; 363, 409, and 476; 363, 464, or 476; 409, 464, and476.

The combination of three substitutions is A272S, Q287K, and S325D;A272S, Q287K, and L347I; A272S, Q287K, and D357N; A272S, Q287K, andS363K; A272S, Q287K, and G409C; A272S, Q287K, and T464Q; A272S, Q287K,and N476C; A272S, S325D, and L347I; A272S, S325D, and D357N; A272S,S325D, and S363K; A272S, S325D, and G409C; A272S, S325D, and T464Q;A272S, S325D, and N476C; A272S, L347I, and D357N; A272S, L347I, andS363K; A272S, L347I, and G409C; A272S, L347I, and T464Q; A272S, L347I,and N476C; A272S, D357N, and S363K; A272S, D357N, and G409C; A272S,D357N, and T464Q; A272S, D357N, and N476C; A272S, S363K, and G409C;A272S, S363K, and T464Q; A272S, S363K, and N476C; A272S, G409C, andT464Q; A272S, G409C, and N476C; A272S, T464Q, and N476C; Q287K, S325D,and L347I; Q287K, S325D, and D357N; Q287K, S325D, and S363K; Q287K,S325D, and G409C; Q287K, S325D, and T464Q; Q287K, S325D, and N476C;Q287K, L347I, and D357N; Q287K, L347I, and S363K; Q287K, L347I, andG409C; Q287K, L347I, and T464Q; Q287K, L347I, and N476C; Q287K, D357N,and S363K; Q287K, D357N, and G409C; Q287K, D357N, and T464Q; Q287K,D357N, and N476C; Q287K, S363K, and G409C; Q287K, S363K, and T464Q;Q287K, S363K, and N476C; Q287K, G409C, and T464Q; Q287K, G409C, andN476C; Q287K, T464Q, and N476C; S325D, L347I, and D357N; S325D, L347I,and S363K; S325D, L347I, and G409C; S325D, L347I, and T464Q; S325D,L347I, and N476C; S325D, D357N, and S363K; S325D, D357N, and G409C;S325D, D357N, and T464Q; S325D, D357N, and N476C; S325D, S363K, andG409C; S325D, S363K, and T464Q; S325D, S363K, and N476C; S325D, G409C,and T464Q; S325D, G409C, and N476C; S325D, T464Q, and N476C; L347I,D357N, and S363K; L347I, D357N, and G409C; L347I, D357N, and T464Q;L347I, D357N, and N476C; L347I, S363K, and G409C; L347I, S363K, andT464Q; L347I, S363K, and N476C; L347I, G409C, and T464Q; L347I, G409C,and N476C; L347I, T464Q, and N476C; D357N, S363K, and G409C; D357N,S363K, and T464Q; D357N, S363K, and N476C; D357N, G409C, and T464Q;D357N, G409C, and N476C; D357N, T464Q, and N476C; S363K, G409C, andT464Q; S363K, G409C, and N476C; S363K, T464Q, and N476C; or G409C,T464Q, and N476C.

In another aspect, the variant comprises or consists of a combination offour substitutions at positions corresponding to any of positions 272,287, 325, 347, 357, 363, 464, 409, and 476 of the mature polypeptide ofSEQ ID NO: 2. In another aspect, the variant comprises or consists of acombination of four substitutions at positions corresponding to any ofpositions 272, 287, 325, 347, 357, 363, 464, 409, and 476 of the maturepolypeptide of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. Inanother aspect, the variant comprises or consists of a combination offour substitutions of any of Ser, Lys, Asp, Ile, Asn, Lys, Gin, Cys, andCys at positions corresponding to positions 272, 287, 325, 347, 357,363, 464, 409, and 476, respectively, of the mature polypeptide of SEQID NO: 2. In another aspect, the variant comprises or consists of acombination of four substitutions of any of A272S, Q287K, S325D, L347I,D357N, S363K, T464Q, G409C, and N476C of the mature polypeptide of SEQID NO: 2.

The combination of four positions is positions 272, 287, 325, and 347;272, 287, 325, and 357; 272, 287, 325, and 363; 272, 287, 325, and 409;272, 287, 325, and 464; 272, 287, 325, and 476; 272, 287, 347, and 357;272, 287, 347, and 363; 272, 287, 347, and 409; 272, 287, 347, and 464;272, 287, 347, and 476; 272, 287, 357, and 363; 272, 287, 357, and 409;272, 287, 357, and 464; 272, 287, 357, and 476; 272, 287, 363, and 409;272, 287, 363, and 464; 272, 287, 363, and 476; 272, 287, 409, and 464;272, 287, 409, and 476; 272, 287, 464, and 476; 272, 325, 347, and 357;272, 325, 347, and 363; 272, 325, 347, and 409; 272, 325, 347, and 464;272, 325, 347, and 476; 272, 325, 357, and 363; 272, 325, 357, and 409;272, 325, 357, and 464; 272, 325, 357, and 476; 272, 325, 363, and 409;272, 325, 363, and 464; 272, 325, 363, and 476; 272, 325, 409, and 464;272, 325, 409, and 476; 272, 325, 464, and 476; 272, 347, 357, and 363;272, 347, 357, and 409; 272, 347, 357, and 464; 272, 347, 357, and 476;272, 347, 363, and 409; 272, 347, 363, and 464; 272, 347, 363, and 476;272, 347, 409, and 464; 272, 347, 409, and 476; 272, 347, 464, and 476;272, 357, 363, and 409; 272, 357, 363, and 464; 272, 357, 363, and 476;272, 357, 409, and 464; 272, 357, 409, and 476; 272, 357, 464, and 476;272, 363, 409, and 464; 272, 363, 409, and 476; 272, 363, 464, and 476;272, 409, 464, and 476; 287, 325, 347, and 357; 287, 325, 347, and 363;287, 325, 347, and 409; 287, 325, 347, and 464; 287, 325, 347, and 476;287, 325, 357, and 363; 287, 325, 357, and 409; 287, 325, 357, and 464;287, 325, 357, and 476; 287, 325, 363, and 409; 287, 325, 363, and 464;287, 325, 363, and 476; 287, 325, 409, and 464; 287, 325, 409, and 476;287, 325, 464, and 476; 287, 347, 357, and 363; 287, 347, 357, and 409;287, 347, 357, and 464; 287, 347, 357, and 476; 287, 347, 363, and 409;287, 347, 363, and 464; 287, 347, 363, and 476; 287, 347, 409, and 464;287, 347, 409, and 476; 287, 347, 464, and 476; 287, 357, 363, and 409;287, 357, 363, and 464; 287, 357, 363, and 476; 287, 357, 409, and 464;287, 357, 409, and 476; 287, 357, 464, and 476; 287, 363, 409, and 464;287, 363, 409, and 476; 287, 363, 464, and 476; 287, 409, 464, and 476;325, 347, 357, and 363; 325, 347, 357, and 409; 325, 347, 357, and 464;325, 347, 357, and 476; 325, 347, 363, and 409; 325, 347, 363, and 464;325, 347, 363, and 476; 325, 347, 409, and 464; 325, 347, 409, and 476;325, 347, 464, and 476; 325, 357, 363, and 409; 325, 357, 363, and 464;325, 357, 363, and 476; 325, 357, 409, and 464; 325, 357, 409, and 476;325, 357, 464, and 476; 325, 363, 409, and 464; 325, 363, 409, and 476;325, 363, 464, and 476; 325, 409, 464, and 476; 347, 357, 363, and 409;347, 357, 363, and 464; 347, 357, 363, and 476; 347, 357, 409, and 464;347, 357, 409, and 476; 347, 357, 464, and 476; 347, 363, 409, and 464;347, 363, 409, and 476; 347, 363, 464, and 476; 347, 409, 464, and 476;357, 363, 409, and 464; 357, 363, 409, and 476; 357, 363, 464, and 476;357, 409, 464, and 476; or 363, 409, 464, and 476.

The combination of four substitutions is A272S, Q287K, S325D, and L347I;A272S, Q287K, S325D, and D357N; A272S, Q287K, S325D, and S363K; A272S,Q287K, S325D, and G409C; A272S, Q287K, S325D, and T464Q; A272S, Q287K,S325D, and N476C; A272S, Q287K, L347I, and D357N; A272S, Q287K, L347I,and S363K; A272S, Q287K, L347I, and G409C; A272S, Q287K, L347I, andT464Q; A272S, Q287K, L347I, and N476C; A272S, Q287K, D357N, and S363K;A272S, Q287K, D357N, and G409C; A272S, Q287K, D357N, and T464Q; A272S,Q287K, D357N, and N476C; A272S, Q287K, S363K, and G409C; A272S, Q287K,S363K, and T464Q; A272S, Q287K, S363K, and N476C; A272S, Q287K, G409C,and T464Q; A272S, Q287K, G409C, and N476C; A272S, Q287K, T464Q, andN476C; A272S, S325D, L347I, and D357N; A272S, S325D, L347I, and S363K;A272S, S325D, L347I, and G409C; A272S, S325D, L347I, and T464Q; A272S,S325D, L347I, and N476C; A272S, S325D, D357N, and S363K; A272S, S325D,D357N, and G409C; A272S, S325D, D357N, and T464Q; A272S, S325D, D357N,and N476C; A272S, S325D, S363K, and G409C; A272S, S325D, S363K, andT464Q; A272S, S325D, S363K, and N476C; A272S, S325D, G409C, and T464Q;A272S, S325D, G409C, and N476C; A272S, S325D, T464Q, and N476C; A272S,L347I, D357N, and S363K; A272S, L347I, D357N, and G409C; A272S, L347I,D357N, and T464Q; A272S, L347I, D357N, and N476C; A272S, L347I, S363K,and G409C; A272S, L347I, S363K, and T464Q; A272S, L347I, S363K, andN476C; A272S, L347I, G409C, and T464Q; A272S, L347I, G409C, and N476C;A272S, L347I, T464Q, and N476C; A272S, D357N, S363K, and G409C; A272S,D357N, S363K, and T464Q; A272S, D357N, S363K, and N476C; A272S, D357N,G409C, and T464Q; A272S, D357N, G409C, and N476C; A272S, D357N, T464Q,and N476C; A272S, S363K, G409C, and T464Q; A272S, S363K, G409C, andN476C; A272S, S363K, T464Q, and N476C; A272S, G409C, T464Q, and N476C;Q287K, S325D, L347I, and D357N; Q287K, S325D, L347I, and S363K; Q287K,S325D, L347I, and G409C; Q287K, S325D, L347I, and T464Q; Q287K, S325D,L347I, and N476C; Q287K, S325D, D357N, and S363K; Q287K, S325D, D357N,and G409C; Q287K, S325D, D357N, and T464Q; Q287K, S325D, D357N, andN476C; Q287K, S325D, S363K, and G409C; Q287K, S325D, S363K, and T464Q;Q287K, S325D, S363K, and N476C; Q287K, S325D, G409C, and T464Q; Q287K,S325D, G409C, and N476C; Q287K, S325D, T464Q, and N476C; Q287K, L347I,D357N, and S363K; Q287K, L347I, D357N, and G409C; Q287K, L347I, D357N,and T464Q; Q287K, L347I, D357N, and N476C; Q287K, L347I, S363K, andG409C; Q287K, L347I, S363K, and T464Q; Q287K, L347I, S363K, and N476C;Q287K, L347I, G409C, and T464Q; Q287K, L347I, G409C, and N476C; Q287K,L347I, T464Q, and N476C; Q287K, D357N, S363K, and G409C; Q287K, D357N,S363K, and T464Q; Q287K, D357N, S363K, and N476C; Q287K, D357N, G409C,and T464Q; Q287K, D357N, G409C, and N476C; Q287K, D357N, T464Q, andN476C; Q287K, S363K, G409C, and T464Q; Q287K, S363K, G409C, and N476C;Q287K, S363K, T464Q, and N476C; Q287K, G409C, T464Q, and N476C; S325D,L347I, D357N, and S363K; S325D, L347I, D357N, and G409C; S325D, L347I,D357N, and T464Q; S325D, L347I, D357N, and N476C; S325D, L347I, S363K,and G409C; S325D, L347I, S363K, and T464Q; S325D, L347I, S363K, andN476C; S325D, L347I, G409C, and T464Q; S325D, L347I, G409C, and N476C;S325D, L347I, T464Q, and N476C; S325D, D357N, S363K, and G409C; S325D,D357N, S363K, and T464Q; S325D, D357N, S363K, and N476C; S325D, D357N,G409C, and T464Q; S325D, D357N, G409C, and N476C; S325D, D357N, T464Q,and N476C; S325D, S363K, G409C, and T464Q; S325D, S363K, G409C, andN476C; S325D, S363K, T464Q, and N476C; S325D, G409C, T464Q, and N476C;L347I, D357N, S363K, and G409C; L347I, D357N, S363K, and T464Q; L347I,D357N, S363K, and N476C; L347I, D357N, G409C, and T464Q; L347I, D357N,G409C, and N476C; L347I, D357N, T464Q, and N476C; L347I, S363K, G409C,and T464Q; L347I, S363K, G409C, and N476C; L347I, S363K, T464Q, andN476C; L347I, G409C, T464Q, and N476C; D357N, S363K, G409C, and T464Q;D357N, S363K, G409C, and N476C; D357N, S363K, T464Q, and N476C; D357N,G409C, T464Q, or N476C; S363K, G409C, T464Q, and N476C.

In another aspect, the variant comprises or consists of a combination offive substitutions at positions corresponding to any of positions 272,287, 325, 347, 357, 363, 464, 409, and 476 of the mature polypeptide ofSEQ ID NO: 2. In another aspect, the variant comprises or consists of acombination of five substitutions at positions corresponding to any ofpositions 272, 287, 325, 347, 357, 363, 464, 409, and 476 of the maturepolypeptide of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. Inanother aspect, the variant comprises or consists of a combination offive substitutions of any of Ser, Lys, Asp, Ile, Asn, Lys, Gln, Cys, andCys at positions corresponding to positions 272, 287, 325, 347, 357,363, 464, 409, and 476, respectively, of the mature polypeptide of SEQID NO: 2. In another aspect, the variant comprises or consists of acombination of five substitutions of any of A272S, Q287K, S325D, L347I,D357N, S363K, T464Q, G409C, and N476C of the mature polypeptide of SEQID NO: 2.

The combination of five positions is positions 272, 287, 325, 347, and357; 272, 287, 325, 347, and 363; 272, 287, 325, 347, and 409; 272, 287,325, 347, and 464; 272, 287, 325, 347, and 476; 272, 287, 325, 357, and363; 272, 287, 325, 357, and 409; 272, 287, 325, 357, and 464; 272, 287,325, 357, and 476; 272, 287, 325, 363, and 409; 272, 287, 325, 363, and464; 272, 287, 325, 363, and 476; 272, 287, 325, 409, and 464; 272, 287,325, 409, and 476; 272, 287, 325, 464, and 476; 272, 287, 347, 357, and363; 272, 287, 347, 357, and 409; 272, 287, 347, 357, and 464; 272, 287,347, 357, and 476; 272, 287, 347, 363, and 409; 272, 287, 347, 363, and464; 272, 287, 347, 363, and 476; 272, 287, 347, 409, and 464; 272, 287,347, 409, and 476; 272, 287, 347, 464, and 476; 272, 287, 357, 363, and409; 272, 287, 357, 363, and 464; 272, 287, 357, 363, and 476; 272, 287,357, 409, and 464; 272, 287, 357, 409, and 476; 272, 287, 357, 464, and476; 272, 287, 363, 409, and 464; 272, 287, 363, 409, and 476; 272, 287,363, 464, and 476; 272, 287, 409, 464, and 476; 272, 325, 347, 357, and363; 272, 325, 347, 357, and 409; 272, 325, 347, 357, and 464; 272, 325,347, 357, and 476; 272, 325, 347, 363, and 409; 272, 325, 347, 363, and464; 272, 325, 347, 363, and 476; 272, 325, 347, 409, and 464; 272, 325,347, 409, and 476; 272, 325, 347, 464, and 476; 272, 325, 357, 363, and409; 272, 325, 357, 363, and 464; 272, 325, 357, 363, and 476; 272, 325,357, 409, and 464; 272, 325, 357, 409, and 476; 272, 325, 357, 464, and476; 272, 325, 363, 409, and 464; 272, 325, 363, 409, and 476; 272, 325,363, 464, and 476; 272, 325, 409, 464, and 476; 272, 347, 357, 363, and409; 272, 347, 357, 363, and 464; 272, 347, 357, 363, and 476; 272, 347,357, 409, and 464; 272, 347, 357, 409, and 476; 272, 347, 357, 464, and476; 272, 347, 363, 409, and 464; 272, 347, 363, 409, and 476; 272, 347,363, 464, and 476; 272, 347, 409, 464, and 476; 272, 357, 363, 409, and464; 272, 357, 363, 409, and 476; 272, 357, 363, 464, and 476; 272, 357,409, 464, and 476; 272, 363, 409, 464, and 476; 287, 325, 347, 357, and363; 287, 325, 347, 357, and 409; 287, 325, 347, 357, and 464; 287, 325,347, 357, and 476; 287, 325, 347, 363, and 409; 287, 325, 347, 363, and464; 287, 325, 347, 363, and 476; 287, 325, 347, 409, and 464; 287, 325,347, 409, and 476; 287, 325, 347, 464, and 476; 287, 325, 357, 363, and409; 287, 325, 357, 363, and 464; 287, 325, 357, 363, and 476; 287, 325,357, 409, and 464; 287, 325, 357, 409, and 476; 287, 325, 357, 464, and476; 287, 325, 363, 409, and 464; 287, 325, 363, 409, and 476; 287, 325,363, 464, and 476; 287, 325, 409, 464, and 476; 287, 347, 357, 363, and409; 287, 347, 357, 363, and 464; 287, 347, 357, 363, and 476; 287, 347,357, 409, and 464; 287, 347, 357, 409, and 476; 287, 347, 357, 464, and476; 287, 347, 363, 409, and 464; 287, 347, 363, 409, and 476; 287, 347,363, 464, and 476; 287, 347, 409, 464, and 476; 287, 357, 363, 409, and464; 287, 357, 363, 409, and 476; 287, 357, 363, 464, and 476; 287, 357,409, 464, and 476; 287, 363, 409, 464, and 476; 325, 347, 357, 363, and409; 325, 347, 357, 363, and 464; 325, 347, 357, 363, and 476; 325, 347,357, 409, and 464; 325, 347, 357, 409, and 476; 325, 347, 357, 464, and476; 325, 347, 363, 409, and 464; 325, 347, 363, 409, and 476; 325, 347,363, 464, and 476; 325, 347, 409, 464, and 476; 325, 357, 363, 409, and464; 325, 357, 363, 409, and 476; 325, 357, 363, 464, and 476; 325, 357,409, 464, and 476; 325, 363, 409, 464, and 476; 347, 357, 363, 409, and464; 347, 357, 363, 409, and 476; 347, 357, 363, 464, and 476; 347, 357,409, 464, and 476; 347, 363, 409, 464, or 476; or 357, 363, 409, 464,and 476.

The combination of five substitutions is A272S, Q287K, S325D, L347I, andD357N; A272S, Q287K, S325D, L347I, and S363K; A272S, Q287K, S325D,L347I, and G409C; A272S, Q287K, S325D, L347I, and T464Q; A272S, Q287K,S325D, L347I, and N476C; A272S, Q287K, S325D, D357N, and S363K; A272S,Q287K, S325D, D357N, and G409C; A272S, Q287K, S325D, D357N, and T464Q;A272S, Q287K, S325D, D357N, and N476C; A272S, Q287K, S325D, S363K, andG409C; A272S, Q287K, S325D, S363K, and T464Q; A272S, Q287K, S325D,S363K, and N476C; A272S, Q287K, S325D, G409C, and T464Q; A272S, Q287K,S325D, G409C, and N476C; A272S, Q287K, S325D, T464Q, and N476C; A272S,Q287K, L347I, D357N, and S363K; A272S, Q287K, L347I, D357N, and G409C;A272S, Q287K, L347I, D357N, and T464Q; A272S, Q287K, L347I, D357N, andN476C; A272S, Q287K, L347I, S363K, and G409C; A272S, Q287K, L347I,S363K, and T464Q; A272S, Q287K, L347I, S363K, and N476C; A272S, Q287K,L347I, G409C, and T464Q; A272S, Q287K, L347I, G409C, and N476C; A272S,Q287K, L347I, T464Q, and N476C; A272S, Q287K, D357N, S363K, and G409C;A272S, Q287K, D357N, S363K, and T464Q; A272S, Q287K, D357N, S363K, andN476C; A272S, Q287K, D357N, G409C, and T464Q; A272S, Q287K, D357N,G409C, and N476C; A272S, Q287K, D357N, T464Q, and N476C; A272S, Q287K,S363K, G409C, and T464Q; A272S, Q287K, S363K, G409C, and N476C; A272S,Q287K, S363K, T464Q, and N476C; A272S, Q287K, G409C, T464Q, and N476C;A272S, S325D, L347I, D357N, and S363K; A272S, S325D, L347I, D357N, andG409C; A272S, S325D, L347I, D357N, and T464Q; A272S, S325D, L347I,D357N, and N476C; A272S, S325D, L347I, S363K, and G409C; A272S, S325D,L347I, S363K, and T464Q; A272S, S325D, L347I, S363K, and N476C; A272S,S325D, L347I, G409C, and T464Q; A272S, S325D, L347I, G409C, and N476C;A272S, S325D, L347I, T464Q, and N476C; A272S, S325D, D357N, S363K, andG409C; A272S, S325D, D357N, S363K, and T464Q; A272S, S325D, D357N,S363K, and N476C; A272S, S325D, D357N, G409C, and T464Q; A272S, S325D,D357N, G409C, and N476C; A272S, S325D, D357N, T464Q, and N476C; A272S,S325D, S363K, G409C, and T464Q; A272S, S325D, S363K, G409C, and N476C;A272S, S325D, S363K, T464Q, and N476C; A272S, S325D, G409C, T464Q, andN476C; A272S, L347I, D357N, S363K, and G409C; A272S, L347I, D357N,S363K, and T464Q; A272S, L347I, D357N, S363K, and N476C; A272S, L347I,D357N, G409C, and T464Q; A272S, L347I, D357N, G409C, and N476C; A272S,L347I, D357N, T464Q, and N476C; A272S, L347I, S363K, G409C, and T464Q;A272S, L347I, S363K, G409C, and N476C; A272S, L347I, S363K, T464Q, andN476C; A272S, L347I, G409C, T464Q, and N476C; A272S, D357N, S363K,G409C, and T464Q; A272S, D357N, S363K, G409C, and N476C; A272S, D357N,S363K, T464Q, and N476C; A272S, D357N, G409C, T464Q, and N476C; A272S,S363K, G409C, T464Q, and N476C; Q287K, S325D, L347I, D357N, and S363K;Q287K, S325D, L347I, D357N, and G409C; Q287K, S325D, L347I, D357N, andT464Q; Q287K, S325D, L347I, D357N, and N476C; Q287K, S325D, L347I,S363K, and G409C; Q287K, S325D, L347I, S363K, and T464Q; Q287K, S325D,L347I, S363K, and N476C; Q287K, S325D, L347I, G409C, and T464Q; Q287K,S325D, L347I, G409C, and N476C; Q287K, S325D, L347I, T464Q, and N476C;Q287K, S325D, D357N, S363K, and G409C; Q287K, S325D, D357N, S363K, andT464Q; Q287K, S325D, D357N, S363K, and N476C; Q287K, S325D, D357N,G409C, and T464Q; Q287K, S325D, D357N, G409C, and N476C; Q287K, S325D,D357N, T464Q, and N476C; Q287K, S325D, S363K, G409C, and T464Q; Q287K,S325D, S363K, G409C, and N476C; Q287K, S325D, S363K, T464Q, and N476C;Q287K, S325D, G409C, T464Q, and N476C; Q287K, L347I, D357N, S363K, andG409C; Q287K, L347I, D357N, S363K, and T464Q; Q287K, L347I, D357N,S363K, and N476C; Q287K, L347I, D357N, G409C, and T464Q; Q287K, L347I,D357N, G409C, and N476C; Q287K, L347I, D357N, T464Q, and N476C; Q287K,L347I, S363K, G409C, and T464Q; Q287K, L347I, S363K, G409C, and N476C;Q287K, L347I, S363K, T464Q, and N476C; Q287K, L347I, G409C, T464Q, andN476C; Q287K, D357N, S363K, G409C, and T464Q; Q287K, D357N, S363K,G409C, and N476C; Q287K, D357N, S363K, T464Q, and N476C; Q287K, D357N,G409C, T464Q, and N476C; Q287K, S363K, G409C, T464Q, and N476C; S325D,L347I, D357N, S363K, and G409C; S325D, L347I, D357N, S363K, and T464Q;S325D, L347I, D357N, S363K, and N476C; S325D, L347I, D357N, G409C, andT464Q; S325D, L347I, D357N, G409C, and N476C; S325D, L347I, D357N,T464Q, and N476C; S325D, L347I, S363K, G409C, and T464Q; S325D, L347I,S363K, G409C, and N476C; S325D, L347I, S363K, T464Q, and N476C; S325D,L347I, G409C, T464Q, and N476C; S325D, D357N, S363K, G409C, and T464Q;S325D, D357N, S363K, G409C, and N476C; S325D, D357N, S363K, T464Q, andN476C; S325D, D357N, G409C, T464Q, and N476C; S325D, S363K, G409C,T464Q, and N476C; L347I, D357N, S363K, G409C, and T464Q; L347I, D357N,S363K, G409C, and N476C; L347I, D357N, S363K, T464Q, and N476C; L347I,D357N, G409C, T464Q, and N476C; L347I, S363K, G409C, T464Q, or N476C;D357N, S363K, G409C, T464Q, and N476C.

In another aspect, the variant comprises or consists of a combination ofsix substitutions at positions corresponding to any of positions 272,287, 325, 347, 357, 363, 464, 409, and 476 of the mature polypeptide ofSEQ ID NO: 2. In another aspect, the variant comprises or consists of acombination of six substitutions at positions corresponding to any ofpositions 272, 287, 325, 347, 357, 363, 464, 409, and 476 of the maturepolypeptide of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. Inanother aspect, the variant comprises or consists of a combination ofsix substitutions of any of Ser, Lys, Asp, Ile, Asn, Lys, Gin, Cys, andCys at positions corresponding to positions 272, 287, 325, 347, 357,363, 464, 409, and 476, respectively, of the mature polypeptide of SEQID NO: 2. In another aspect, the variant comprises or consists of acombination of six substitutions of any of A272S, Q287K, S325D, L347I,D357N, S363K, T464Q, G409C, and N476C of the mature polypeptide of SEQID NO: 2.

The combination of six positions is positions 272, 287, 325, 347, 357,and 363; 272, 287, 325, 347, 357, and 409; 272, 287, 325, 347, 357, and464; 272, 287, 325, 347, 357, and 476; 272, 287, 325, 347, 363, and 409;272, 287, 325, 347, 363, and 464; 272, 287, 325, 347, 363, and 476; 272,287, 325, 347, 409, and 464; 272, 287, 325, 347, 409, and 476; 272, 287,325, 347, 464, and 476; 272, 287, 325, 357, 363, and 409; 272, 287, 325,357, 363, and 464; 272, 287, 325, 357, 363, and 476; 272, 287, 325, 357,409, and 464; 272, 287, 325, 357, 409, and 476; 272, 287, 325, 357, 464,and 476; 272, 287, 325, 363, 409, and 464; 272, 287, 325, 363, 409, and476; 272, 287, 325, 363, 464, and 476; 272, 287, 325, 409, 464, and 476;272, 287, 347, 357, 363, and 409; 272, 287, 347, 357, 363, and 464; 272,287, 347, 357, 363, and 476; 272, 287, 347, 357, 409, and 464; 272, 287,347, 357, 409, and 476; 272, 287, 347, 357, 464, and 476; 272, 287, 347,363, 409, and 464; 272, 287, 347, 363, 409, and 476; 272, 287, 347, 363,464, and 476; 272, 287, 347, 409, 464, and 476; 272, 287, 357, 363, 409,and 464; 272, 287, 357, 363, 409, and 476; 272, 287, 357, 363, 464, and476; 272, 287, 357, 409, 464, and 476; 272, 287, 363, 409, 464, and 476;272, 325, 347, 357, 363, and 409; 272, 325, 347, 357, 363, and 464; 272,325, 347, 357, 363, and 476; 272, 325, 347, 357, 409, and 464; 272, 325,347, 357, 409, and 476; 272, 325, 347, 357, 464, and 476; 272, 325, 347,363, 409, and 464; 272, 325, 347, 363, 409, and 476; 272, 325, 347, 363,464, and 476; 272, 325, 347, 409, 464, and 476; 272, 325, 357, 363, 409,and 464; 272, 325, 357, 363, 409, and 476; 272, 325, 357, 363, 464, and476; 272, 325, 357, 409, 464, and 476; 272, 325, 363, 409, 464, and 476;272, 347, 357, 363, 409, and 464; 272, 347, 357, 363, 409, and 476; 272,347, 357, 363, 464, and 476; 272, 347, 357, 409, 464, and 476; 272, 347,363, 409, 464, and 476; 272, 357, 363, 409, 464, and 476; 287, 325, 347,357, 363, and 409; 287, 325, 347, 357, 363, and 464; 287, 325, 347, 357,363, and 476; 287, 325, 347, 357, 409, and 464; 287, 325, 347, 357, 409,and 476; 287, 325, 347, 357, 464, and 476; 287, 325, 347, 363, 409, and464; 287, 325, 347, 363, 409, and 476; 287, 325, 347, 363, 464, and 476;287, 325, 347, 409, 464, and 476; 287, 325, 357, 363, 409, and 464; 287,325, 357, 363, 409, and 476; 287, 325, 357, 363, 464, and 476; 287, 325,357, 409, 464, and 476; 287, 325, 363, 409, 464, and 476; 287, 347, 357,363, 409, and 464; 287, 347, 357, 363, 409, and 476; 287, 347, 357, 363,464, and 476; 287, 347, 357, 409, 464, and 476; 287, 347, 363, 409, 464,and 476; 287, 357, 363, 409, 464, and 476; 325, 347, 357, 363, 409, and464; 325, 347, 357, 363, 409, and 476; 325, 347, 357, 363, 464, and 476;325, 347, 357, 409, 464, and 476; 325, 347, 363, 409, 464, and 476; 325,357, 363, 409, 464, and 476; or 347, 357, 363, 409, 464, and 476.

The combination of six substitutions is A272S, Q287K, S325D, L347I,D357N, and S363K; A272S, Q287K, S325D, L347I, D357N, and G409C; A272S,Q287K, S325D, L347I, D357N, and T464Q; A272S, Q287K, S325D, L347I,D357N, and N476C; A272S, Q287K, S325D, L347I, S363K, and G409C; A272S,Q287K, S325D, L347I, S363K, and T464Q; A272S, Q287K, S325D, L347I,S363K, and N476C; A272S, Q287K, S325D, L347I, G409C, and T464Q; A272S,Q287K, S325D, L347I, G409C, and N476C; A272S, Q287K, S325D, L347I,T464Q, and N476C; A272S, Q287K, S325D, D357N, S363K, and G409C; A272S,Q287K, S325D, D357N, S363K, and T464Q; A272S, Q287K, S325D, D357N,S363K, and N476C; A272S, Q287K, S325D, D357N, G409C, and T464Q; A272S,Q287K, S325D, D357N, G409C, and N476C; A272S, Q287K, S325D, D357N,T464Q, and N476C; A272S, Q287K, S325D, S363K, G409C, and T464Q; A272S,Q287K, S325D, S363K, G409C, and N476C; A272S, Q287K, S325D, S363K,T464Q, and N476C; A272S, Q287K, S325D, G409C, T464Q, and N476C; A272S,Q287K, L347I, D357N, S363K, and G409C; A272S, Q287K, L347I, D357N,S363K, and T464Q; A272S, Q287K, L347I, D357N, S363K, and N476C; A272S,Q287K, L347I, D357N, G409C, and T464Q; A272S, Q287K, L347I, D357N,G409C, and N476C; A272S, Q287K, L347I, D357N, T464Q, and N476C; A272S,Q287K, L347I, S363K, G409C, and T464Q; A272S, Q287K, L347I, S363K,G409C, and N476C; A272S, Q287K, L347I, S363K, T464Q, and N476C; A272S,Q287K, L347I, G409C, T464Q, and N476C; A272S, Q287K, D357N, S363K,G409C, and T464Q; A272S, Q287K, D357N, S363K, G409C, and N476C; A272S,Q287K, D357N, S363K, T464Q, and N476C; A272S, Q287K, D357N, G409C,T464Q, and N476C; A272S, Q287K, S363K, G409C, T464Q, and N476C; A272S,S325D, L347I, D357N, S363K, and G409C; A272S, S325D, L347I, D357N,S363K, and T464Q; A272S, S325D, L347I, D357N, S363K, and N476C; A272S,S325D, L347I, D357N, G409C, and T464Q; A272S, S325D, L347I, D357N,G409C, and N476C; A272S, S325D, L347I, D357N, T464Q, and N476C; A272S,S325D, L347I, S363K, G409C, and T464Q; A272S, S325D, L347I, S363K,G409C, and N476C; A272S, S325D, L347I, S363K, T464Q, and N476C; A272S,S325D, L347I, G409C, T464Q, and N476C; A272S, S325D, D357N, S363K,G409C, and T464Q; A272S, S325D, D357N, S363K, G409C, and N476C; A272S,S325D, D357N, S363K, T464Q, and N476C; A272S, S325D, D357N, G409C,T464Q, and N476C; A272S, S325D, S363K, G409C, T464Q, and N476C; A272S,L347I, D357N, S363K, G409C, and T464Q; A272S, L347I, D357N, S363K,G409C, and N476C; A272S, L347I, D357N, S363K, T464Q, and N476C; A272S,L347I, D357N, G409C, T464Q, and N476C; A272S, L347I, S363K, G409C,T464Q, and N476C; A272S, D357N, S363K, G409C, T464Q, and N476C; Q287K,S325D, L347I, D357N, S363K, and G409C; Q287K, S325D, L347I, D357N,S363K, and T464Q; Q287K, S325D, L347I, D357N, S363K, and N476C; Q287K,S325D, L347I, D357N, G409C, and T464Q; Q287K, S325D, L347I, D357N,G409C, and N476C; Q287K, S325D, L347I, D357N, T464Q, and N476C; Q287K,S325D, L347I, S363K, G409C, and T464Q; Q287K, S325D, L347I, S363K,G409C, and N476C; Q287K, S325D, L347I, S363K, T464Q, and N476C; Q287K,S325D, L347I, G409C, T464Q, and N476C; Q287K, S325D, D357N, S363K,G409C, and T464Q; Q287K, S325D, D357N, S363K, G409C, and N476C; Q287K,S325D, D357N, S363K, T464Q, and N476C; Q287K, S325D, D357N, G409C,T464Q, and N476C; Q287K, S325D, S363K, G409C, T464Q, and N476C; Q287K,L347I, D357N, S363K, G409C, and T464Q; Q287K, L347I, D357N, S363K,G409C, and N476C; Q287K, L347I, D357N, S363K, T464Q, and N476C; Q287K,L347I, D357N, G409C, T464Q, and N476C; Q287K, L347I, S363K, G409C,T464Q, and N476C; Q287K, D357N, S363K, G409C, T464Q, and N476C; S325D,L347I, D357N, S363K, G409C, and T464Q; S325D, L347I, D357N, S363K,G409C, and N476C; S325D, L347I, D357N, S363K, T464Q, and N476C; S325D,L347I, D357N, G409C, T464Q, and N476C; S325D, L347I, S363K, G409C,T464Q, and N476C; S325D, D357N, S363K, G409C, T464Q, and N476C; orL347I, D357N, S363K, G409C, T464Q, and N476C.

In another aspect, the variant comprises or consists of a combination ofseven substitutions at positions corresponding to any of positions 272,287, 325, 347, 357, 363, 464, 409, and 476 of the mature polypeptide ofSEQ ID NO: 2. In another aspect, the variant comprises or consists of acombination of seven substitutions at positions corresponding to any ofpositions 272, 287, 325, 347, 357, 363, 464, 409, and 476 of the maturepolypeptide of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. Inanother aspect, the variant comprises or consists of a combination ofseven substitutions of any of Ser, Lys, Asp, Ile, Asn, Lys, Gln, Cys,and Cys at positions corresponding to positions 272, 287, 325, 347, 357,363, 464, 409, and 476, respectively, of the mature polypeptide of SEQID NO: 2. In another aspect, the variant comprises or consists of acombination of seven substitutions of any of A272S, Q287K, S325D, L347I,D357N, S363K, T464Q, G409C, and N476C of the mature polypeptide of SEQID NO: 2.

The combination of seven positions is positions 272, 287, 325, 347, 357,363, and 409; 272, 287, 325, 347, 357, 363, and 464; 272, 287, 325, 347,357, 363, and 476; 272, 287, 325, 347, 357, 409, and 464; 272, 287, 325,347, 357, 409, and 476; 272, 287, 325, 347, 357, 464, and 476; 272, 287,325, 347, 363, 409, and 464; 272, 287, 325, 347, 363, 409, and 476; 272,287, 325, 347, 363, 464, and 476; 272, 287, 325, 347, 409, 464, and 476;272, 287, 325, 357, 363, 409, and 464; 272, 287, 325, 357, 363, 409, and476; 272, 287, 325, 357, 363, 464, and 476; 272, 287, 325, 357, 409,464, and 476; 272, 287, 325, 363, 409, 464, and 476; 272, 287, 347, 357,363, 409, and 464; 272, 287, 347, 357, 363, 409, and 476; 272, 287, 347,357, 363, 464, and 476; 272, 287, 347, 357, 409, 464, and 476; 272, 287,347, 363, 409, 464, and 476; 272, 287, 357, 363, 409, 464, and 476; 272,325, 347, 357, 363, 409, and 464; 272, 325, 347, 357, 363, 409, and 476;272, 325, 347, 357, 363, 464, and 476; 272, 325, 347, 357, 409, 464, and476; 272, 325, 347, 363, 409, 464, and 476; 272, 325, 357, 363, 409,464, and 476; 272, 347, 357, 363, 409, 464, and 476; 287, 325, 347, 357,363, 409, and 464; 287, 325, 347, 357, 363, 409, and 476; 287, 325, 347,357, 363, 464, and 476; 287, 325, 347, 357, 409, 464, and 476; 287, 325,347, 363, 409, 464, and 476; 287, 325, 357, 363, 409, 464, and 476; 287,347, 357, 363, 409, 464, or 476; 325, 347, 357, 363, 409, 464, and 476.

The combination of seven substitutions is A272S, Q287K, S325D, L347I,D357N, S363K, and G409C; A272S, Q287K, S325D, L347I, D357N, S363K, andT464Q; A272S, Q287K, S325D, L347I, D357N, S363K, and N476C; A272S,Q287K, S325D, L347I, D357N, G409C, and T464Q; A272S, Q287K, S325D,L347I, D357N, G409C, and N476C; A272S, Q287K, S325D, L347I, D357N,T464Q, and N476C; A272S, Q287K, S325D, L347I, S363K, G409C, and T464Q;A272S, Q287K, S325D, L347I, S363K, G409C, and N476C; A272S, Q287K,S325D, L347I, S363K, T464Q, and N476C; A272S, Q287K, S325D, L347I,G409C, T464Q, and N476C; A272S, Q287K, S325D, D357N, S363K, G409C, andT464Q; A272S, Q287K, S325D, D357N, S363K, G409C, and N476C; A272S,Q287K, S325D, D357N, S363K, T464Q, and N476C; A272S, Q287K, S325D,D357N, G409C, T464Q, and N476C; A272S, Q287K, S325D, S363K, G409C,T464Q, and N476C; A272S, Q287K, L347I, D357N, S363K, G409C, and T464Q;A272S, Q287K, L347I, D357N, S363K, G409C, and N476C; A272S, Q287K,L347I, D357N, S363K, T464Q, and N476C; A272S, Q287K, L347I, D357N,G409C, T464Q, and N476C; A272S, Q287K, L347I, S363K, G409C, T464Q, andN476C; A272S, Q287K, D357N, S363K, G409C, T464Q, and N476C; A272S,S325D, L347I, D357N, S363K, G409C, and T464Q; A272S, S325D, L347I,D357N, S363K, G409C, and N476C; A272S, S325D, L347I, D357N, S363K,T464Q, and N476C; A272S, S325D, L347I, D357N, G409C, T464Q, and N476C;A272S, S325D, L347I, S363K, G409C, T464Q, and N476C; A272S, S325D,D357N, S363K, G409C, T464Q, and N476C; A272S, L347I, D357N, S363K,G409C, T464Q, and N476C; Q287K, S325D, L347I, D357N, S363K, G409C, andT464Q; Q287K, S325D, L347I, D357N, S363K, G409C, and N476C; Q287K,S325D, L347I, D357N, S363K, T464Q, and N476C; Q287K, S325D, L347I,D357N, G409C, T464Q, and N476C; Q287K, S325D, L347I, S363K, G409C,T464Q, and N476C; Q287K, S325D, D357N, S363K, G409C, T464Q, and N476C;Q287K, L347I, D357N, S363K, G409C, T464Q, and N476C; or S325D, L347I,D357N, S363K, G409C, T464Q, and N476C.

In another aspect, the variant comprises or consists of a combination ofeight substitutions at positions corresponding to any of positions 272,287, 325, 347, 357, 363, 464, 409, and 476 of the mature polypeptide ofSEQ ID NO: 2. In another aspect, the variant comprises or consists of acombination of eight substitutions at positions corresponding to any ofpositions 272, 287, 325, 347, 357, 363, 464, 409, and 476 of the maturepolypeptide of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. Inanother aspect, the variant comprises or consists of a combination ofeight substitutions of any of Ser, Lys, Asp, Ile, Asn, Lys, Gin, Cys,and Cys at positions corresponding to positions 272, 287, 325, 347, 357,363, 464, 409, and 476, respectively, of the mature polypeptide of SEQID NO: 2. In another aspect, the variant comprises or consists of acombination of eight substitutions of any of A272S, Q287K, S325D, L347I,D357N, S363K, T464Q, G409C, and N476C of the mature polypeptide of SEQID NO: 2.

The combination of eight positions is positions 272, 287, 325, 347, 357,363, 409, and 464; 272, 287, 325, 347, 357, 363, 409, and 476; 272, 287,325, 347, 357, 363, 464, and 476; 272, 287, 325, 347, 357, 409, 464, and476; 272, 287, 325, 347, 363, 409, 464, and 476; 272, 287, 325, 357,363, 409, 464, and 476; 272, 287, 347, 357, 363, 409, 464, and 476; 272,325, 347, 357, 363, 409, 464, and 476; or 287, 325, 347, 357, 363, 409,464, and 476.

The combination of eight substitutions is A272S, Q287K, S325D, L347I,D357N, S363K, G409C, and T464Q; A272S, Q287K, S325D, L347I, D357N,S363K, G409C, and N476C; A272S, Q287K, S325D, L347I, D357N, S363K,T464Q, and N476C; A272S, Q287K, S325D, L347I, D357N, G409C, T464Q, andN476C; A272S, Q287K, S325D, L347I, S363K, G409C, T464Q, and N476C;A272S, Q287K, S325D, D357N, S363K, G409C, T464Q, and N476C; A272S,Q287K, L347I, D357N, S363K, G409C, T464Q, and N476C; A272S, S325D,L347I, D357N, S363K, G409C, T464Q, and N476C; or Q287K, S325D, L347I,D357N, S363K, G409C, T464Q, and N476C.

In another aspect, the variant comprises or consists of a combination ofnine substitutions at positions corresponding to positions 272, 287,325, 347, 357, 363, 464, 409, and 476 of the mature polypeptide of SEQID NO: 2. In another aspect, the variant comprises or consists of acombination of nine substitutions at positions corresponding topositions 272, 287, 325, 347, 357, 363, 464, 409, and 476 of the maturepolypeptide of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. Inanother aspect, the variant comprises or consists of a combination ofnine substitutions of Ser, Lys, Asp, Ile, Asn, Lys, Gin, Cys, and Cys atpositions corresponding to positions 272, 287, 325, 347, 357, 363, 464,409, and 476, respectively, of the mature polypeptide of SEQ ID NO: 2.In another aspect, the variant comprises or consists of a combination ofnine substitutions of A272S, Q287K, S325D, L347I, D357N, S363K, G409C,T464Q, and N476C of the mature polypeptide of SEQ ID NO: 2.

The variants of the present invention described above may furthercomprise one or more (several) substitutions, deletions, and/orinsertions of the amino acid sequence.

In one aspect, the variant further comprises a substitution at aposition corresponding to position 435 of the mature polypeptide of SEQID NO: 2. In another aspect, the variant further comprises asubstitution at a position corresponding to position 435 of the maturepolypeptide of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. Inanother aspect, the variant further comprises Ser as a substitution at aposition corresponding to position 435 of the mature polypeptide of SEQID NO: 2. In another aspect, the variant further comprises thesubstitution G435S of the mature polypeptide of SEQ ID NO: 2.

Essential amino acids in a parent can be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations areintroduced at every residue in the molecule, and the resultant mutantmolecules are tested for cellobiohydrolase II activity to identify aminoacid residues that are critical to the activity of the molecule. Seealso, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The activesite of the cellobiohydrolase II or other biological interaction canalso be determined by physical analysis of structure, as determined bysuch techniques as nuclear magnetic resonance, crystallography, electrondiffraction, or photoaffinity labeling, in conjunction with mutation ofputative contact site amino acids. See, for example, de Vos et al.,1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224:899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identities ofessential amino acids can also be inferred from analysis of identitieswith polypeptides that are related to the parent.

The variants may consist of 391 to 400, 401 to 410, 411 to 420, 421 to430, 431 to 440, 441 to 450, or 451 to 460 amino acids.

Polynucleotides

The present invention also relates to isolated polynucleotides thatencode any of the variants of the present invention.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide encoding a variant of the present invention operablylinked to one or more (several) control sequences that direct theexpression of the coding sequence in a suitable host cell underconditions compatible with the control sequences.

The polynucleotide may be manipulated in a variety of ways to providefor expression of a variant. Manipulation of the polynucleotide prior toits insertion into a vector may be desirable or necessary depending onthe expression vector. The techniques for modifying polynucleotidesutilizing recombinant DNA methods are well known in the art.

The control sequence may be a promoter sequence, which is recognized bya host cell for expression of the polynucleotide. The promoter sequencecontains transcriptional control sequences that mediate the expressionof the variant. The promoter may be any nucleic acid sequence that showstranscriptional activity in the host cell including mutant, truncated,and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xylA and xylB genes, E. colilac operon, Streptomyces coelicolor agarase gene (dagA), and prokaryoticbeta-lactamase gene (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci.USA 75: 3727-3731), as well as the tac promoter (DeBoer et al., 1983,Proc. Natl. Acad. Sci. USA 80: 21-25). Further promoters are describedin “Useful proteins from recombinant bacteria” in Gilbert et al., 1980,Scientific American 242: 74-94; and in Sambrook et al., 1989, supra.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are the promoters obtained from the genes for Aspergillusnidulans acetamidase, Aspergillus niger neutral alpha-amylase,Aspergillus niger acid stable alpha-amylase, Aspergillus niger orAspergillus awamori glucoamylase (glaA), Aspergillus oryzae TAKAamylase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triosephosphate isomerase, Fusarium oxysporum trypsin-like protease (WO96/00787), Fusarium venenatum amyloglucosidase (WO 00/56900), Fusariumvenenatum Dania (WO 00/56900), Fusarium venenatum Quinn (WO 00/56900),Rhizomucor miehei lipase, Rhizomucor miehei aspartic proteinase,Trichoderma reesei beta-glucosidase, Trichoderma reeseicellobiohydrolase I, Trichoderma reesei cellobiohydrolase II,Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II,Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanaseIV, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,Trichoderma reesei xylanase II, Trichoderma reesei beta-xylosidase, aswell as the NA2-tpi promoter (a modified promoter including a geneencoding a neutral alpha-amylase in Aspergilli in which the untranslatedleader has been replaced by an untranslated leader from a gene encodingtriose phosphate isomerase in Aspergilli; non-limiting examples includemodified promoters including the gene encoding neutral alpha-amylase inAspergillus niger in which the untranslated leader has been replaced byan untranslated leader from the gene encoding triose phosphate isomerasein Aspergillus nidulans or Aspergillus oryzae); and mutant, truncated,and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a suitable transcription terminatorsequence, which is recognized by a host cell to terminate transcription.The terminator sequence is operably linked to the 3′-terminus of thepolynucleotide encoding the variant. Any terminator that is functionalin the host cell may be used.

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans anthranilate synthase,Aspergillus nigeralpha-glucosidase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-likeprotease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA that is important for translation by thehost cell. The leader sequence is operably linked to the 5′-terminus ofthe polynucleotide encoding the variant. Any leader sequence that isfunctional in the host cell may be used.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the variant-encoding sequence and,when transcribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell may be used.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus nidulans anthranilatesynthase, Aspergillus niger glucoamylase, Aspergillus nigeralpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusariumoxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a variant anddirects the variant into the cell's secretory pathway. The 5′-end of thecoding sequence of the polynucleotide may inherently contain a signalpeptide coding region naturally linked in translation reading frame withthe segment of the coding region that encodes the variant.Alternatively, the 5′-end of the coding sequence may contain a signalpeptide coding region that is foreign to the coding sequence. Theforeign signal peptide coding region may be required where the codingsequence does not naturally contain a signal peptide coding region.Alternatively, the foreign signal peptide coding region may simplyreplace the natural signal peptide coding region in order to enhancesecretion of the variant. However, any signal peptide coding region thatdirects the expressed variant into the secretory pathway of a host cellmay be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCI B 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding region that encodesa propeptide positioned at the N-terminus of a variant. The resultantpolypeptide is known as a proenzyme or propolypeptide (or a zymogen insome cases). A propolypeptide is generally inactive and can be convertedto an active polypeptide by catalytic or autocatalytic cleavage of thepropeptide from the propolypeptide. The propeptide coding region may beobtained from the genes for Bacillus subtilis alkaline protease (aprE),Bacillus subtilis neutral protease (nprT), Myceliophthora thermophilalaccase (WO 95/33836), Rhizomucor miehei aspartic proteinase, andSaccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide regions are present at theN-terminus of a variant, the propeptide region is positioned next to theN-terminus of the variant and the signal peptide region is positionednext to the N-terminus of the propeptide region.

It may also be desirable to add regulatory sequences that allow theregulation of the expression of the variant relative to the growth ofthe host cell. Examples of regulatory systems are those that cause theexpression of the gene to be turned on or off in response to a chemicalor physical stimulus, including the presence of a regulatory compound.Regulatory systems in prokaryotic systems include the lac, tac, and trpoperator systems. In yeast, the ADH2 system or GAL1 system may be used.In filamentous fungi, the Aspergillus niger glucoamylase promoter,Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used. Other examples of regulatorysequences are those that allow for gene amplification. In eukaryoticsystems, these regulatory sequences include the dihydrofolate reductasegene that is amplified in the presence of methotrexate, and themetallothionein genes that are amplified with heavy metals. In thesecases, the polynucleotide encoding the variant would be operably linkedwith the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide encoding a variant of the present invention,a promoter, and transcriptional and translational stop signals. Thevarious nucleotide and control sequences may be joined together toproduce a recombinant expression vector that may include one or more(several) convenient restriction sites to allow for insertion orsubstitution of the polynucleotide encoding the variant at such sites.Alternatively, the polynucleotide may be expressed by inserting thepolynucleotide or a nucleic acid construct comprising the polynucleotideinto an appropriate vector for expression. In creating the expressionvector, the coding sequence is located in the vector so that the codingsequence is operably linked with the appropriate control sequences forexpression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about the expression of the polynucleotide. The choice ofthe vector will typically depend on the compatibility of the vector withthe host cell into which the vector is to be introduced. The vector maybe a linear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more (several) selectable markersthat permit easy selection of transformed, transfected, transduced, orthe like cells. A selectable marker is a gene the product of whichprovides for biocide or viral resistance, resistance to heavy metals,prototrophy to auxotrophs, and the like.

Examples of bacterial selectable markers are the dal genes from Bacilluslicheniformis or Bacillus subtilis, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, ortetracycline resistance. Suitable markers for yeast host cells are ADE2,HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in afilamentous fungal host cell include, but are not limited to, amdS(acetamidase), argB (ornithine carbamoyltransferase), bar(phosphinothricin acetyltransferase), hph (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Preferred for use in an Aspergillus cell are the amdS and pyrG genes ofAspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the variant or any other element ofthe vector for integration into the genome by homologous ornonhomologous recombination. Alternatively, the vector may containadditional nucleotide sequences for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofidentity to the corresponding target sequence to enhance the probabilityof homologous recombination. The integrational elements may be anysequence that is homologous with the target sequence in the genome ofthe host cell. Furthermore, the integrational elements may benon-encoding or encoding nucleotide sequences. On the other hand, thevector may be integrated into the genome of the host cell bynon-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means anucleotide sequence that enables a plasmid or vector to replicate invivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMß1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANSI (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into the host cell to increase production of a variant. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra) to obtain substantially pure variants.

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide encoding a variant of the present invention operablylinked to one or more (several) control sequences that direct theproduction of a variant of the present invention. A construct or vectorcomprising a polynucleotide is introduced into a host cell so that theconstruct or vector is maintained as a chromosomal integrant or as aself-replicating extra-chromosomal vector as described earlier. The term“host cell” encompasses any progeny of a parent cell that is notidentical to the parent cell due to mutations that occur duringreplication. The choice of a host cell will to a large extent dependupon the gene encoding the variant and its source.

The host cell may be any cell useful in the recombinant production of avariant, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any gram-positive or gram-negativebacterium. Gram-positive bacteria include, but are not limited to,Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, andStreptomyces. Gram-negative bacteria include, but are not limited to,Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell, including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell, including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell, including,but not limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may, for instance, beeffected by protoplast transformation (see, e.g., Chang and Cohen, 1979,Mol. Gen. Genet. 168: 111-115), by using competent cells (see, e.g.,Young and Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), by electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or byconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may, forinstance, be effected by protoplast transformation (see, e.g., Hanahan,1983, J. Mol. Biol. 166: 557-580) or electroporation (see, e.g., Doweret al., 1988, Nucleic Acids Res. 16: 6127-6145). The introduction of DNAinto a Streptomyces cell may, for instance, be effected by protoplasttransformation and electroporation (see, e.g., Gong et al., 2004, FoliaMicrobiol. (Praha) 49: 399-405), by conjugation (see, e.g., Mazodier etal., 1989, J. Bacteriol. 171: 3583-3585), or by transduction (see, e.g.,Burke et al., 2001, Proc. Natl. Acad. Sci. USA 98: 6289-6294). Theintroduction of DNA into a Pseudomonas cell may, for instance, beeffected by electroporation (see, e.g., Choi et al., 2006, J. Microbiol.Methods 64: 391-397) or by conjugation (see, e.g., Pinedo and Smets,2005, Appl. Environ. Microbiol. 71: 51-57). The introduction of DNA intoa Streptococcus cell may, for instance, be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), by protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-2070, by electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. Microbiol. 65: 3800-3804) or by conjugation(see, e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, anymethod known in the art for introducing DNA into a host cell can beused.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as wellas the Oomycota and all mitosporic fungi (as defined by Hawksworth etal., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,1995, CAB International, University Press, Cambridge, UK).

The fungal host cell may be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, F. A., Passmore, S. M., andDavenport, R. R., eds, Soc. App. Bacteriol. Symposium Series No. 9,1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporiumzonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsuiphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phiebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238023 and Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474. Suitable methods for transforming Fusarium species aredescribed by Malardier et al., 1989, Gene 78: 147-156, and WO 96/00787.Yeast may be transformed using the procedures described by Becker andGuarente, In Abelson, J. N. and Simon, M. I., editors, Guide to YeastGenetics and Molecular Biology, Methods in Enzymology, Volume 194, pp182-187, Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol.153: 163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a variant,comprising: (a) cultivating a host cell of the present invention underconditions suitable for the expression of the variant; and (b)recovering the variant.

The host cells are cultivated in a nutrient medium suitable forproduction of the variant using methods known in the art. For example,the cell may be cultivated by shake flask cultivation, or small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing the variantto be expressed and/or isolated. The cultivation takes place in asuitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the variant is secreted into the nutrient medium, thevariant can be recovered directly from the medium. If the variant is notsecreted, it can be recovered from cell lysates.

The variant may be detected using methods known in the art that arespecific for the variants. These detection methods may include use ofspecific antibodies, formation of an enzyme product, or disappearance ofan enzyme substrate. For example, an enzyme assay may be used todetermine the activity of the variant.

The variant may be recovered by methods known in the art. For example,the variant may be recovered from the nutrient medium by conventionalprocedures including, but not limited to, collection, centrifugation,filtration, extraction, spray-drying, evaporation, or precipitation.

The variant may be purified by a variety of procedures known in the artincluding, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, J.-C. Jansonand Lars Ryden, editors, VCH Publishers, New York, 1989) to obtainsubstantially pure variants.

In an alternative aspect, the variant is not recovered, but rather ahost cell of the present invention expressing a variant is used as asource of the variant.

Compositions

The present invention also relates to compositions comprising a variantof the present invention.

The composition may comprise a variant of the present invention as themajor enzymatic component, e.g., a mono-component composition.Alternatively, the composition may comprise multiple enzymaticactivities, such as one or more (several) enzymes selected from thegroup consisting of a cellulase, a GH61 polypeptide having cellulolyticenhancing activity, a hemicellulase, an expansin, an esterase, alaccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease,and a swollenin.

The polypeptide compositions may be prepared in accordance with methodsknown in the art and may be in the form of a liquid or a drycomposition. For instance, the polypeptide composition may be in theform of a granulate or a microgranulate. The polypeptide to be includedin the composition may be stabilized in accordance with methods known inthe art.

Examples are given below of preferred uses of the polypeptidecompositions of the invention. The dosage of the polypeptide compositionof the invention and other conditions under which the composition isused may be determined on the basis of methods known in the art.

Uses

The present invention is also directed to the following methods forusing the variants, or compositions thereof.

The present invention also relates to methods for degrading orconverting a cellulosic material, comprising: treating the cellulosicmaterial with an enzyme composition in the presence of a variant of thepresent invention. In one aspect, the method above further comprisesrecovering the degraded or converted cellulosic material. Solubleproducts of degradation or conversion of the cellulosic material can beseparated from the insoluble cellulosic material using technology wellknown in the art such as, for example, centrifugation, filtration, andgravity settling.

The present invention also relates to methods for producing afermentation product, comprising: (a) saccharifying a cellulosicmaterial with an enzyme composition in the presence of a variant of thepresent invention; (b) fermenting the saccharified cellulosic materialwith one or more (several) fermenting microorganisms to produce thefermentation product; and (c) recovering the fermentation product fromthe fermentation.

The present invention also relates to methods of fermenting a cellulosicmaterial, comprising: fermenting the cellulosic material with one ormore (several) fermenting microorganisms, wherein the cellulosicmaterial is saccharified with an enzyme composition in the presence of avariant of the present invention. In one aspect, the fermenting of thecellulosic material produces a fermentation product. In another aspect,the method further comprises recovering the fermentation product fromthe fermentation.

The processing of the cellulosic material according to the presentinvention can be accomplished using processes conventional in the art.Moreover, the methods of the present invention can be implemented usingany conventional biomass processing apparatus configured to operate inaccordance with the invention.

Hydrolysis (saccharification) and fermentation, separate orsimultaneous, include, but are not limited to, separate hydrolysis andfermentation (SHF); simultaneous saccharification and fermentation(SSF); simultaneous saccharification and cofermentation (SSCF); hybridhydrolysis and fermentation (HHF); separate hydrolysis andco-fermentation (SHCF); hybrid hydrolysis and co-fermentation (HHCF);and direct microbial conversion (DMC). SHF uses separate process stepsto first enzymatically hydrolyze cellulosic material to fermentablesugars, e.g., glucose, cellobiose, cellotriose, and pentose sugars, andthen ferment the fermentable sugars to ethanol. In SSF, the enzymatichydrolysis of the cellulosic material and the fermentation of sugars toethanol are combined in one step (Philippidis, G. P., 1996, Cellulosebioconversion technology, in Handbook on Bioethanol: Production andUtilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C.,179-212). SSCF involves the cofermentation of multiple sugars (Sheehan,J., and Himmel, M., 1999, Enzymes, energy and the environment: Astrategic perspective on the U.S. Department of Energy's research anddevelopment activities for bioethanol, Biotechnol. Prog. 15: 817-827).HHF involves a separate hydrolysis step, and in addition a simultaneoussaccharification and hydrolysis step, which can be carried out in thesame reactor. The steps in an HHF process can be carried out atdifferent temperatures, i.e., high temperature enzymaticsaccharification followed by SSF at a lower temperature that thefermentation strain can tolerate. DMC combines all three processes(enzyme production, hydrolysis, and fermentation) in one or more(several) steps where the same organism is used to produce the enzymesfor conversion of the cellulosic material to fermentable sugars and toconvert the fermentable sugars into a final product (Lynd, L. R.,Weimer, P. J., van Zyl, W. H., and Pretorius, I. S., 2002, Microbialcellulose utilization: Fundamentals and biotechnology, Microbiol. Mol.Biol. Reviews 66: 506-577). It is understood herein that any methodknown in the art comprising pretreatment, enzymatic hydrolysis(saccharification), fermentation, or a combination thereof, can be usedin the practicing the methods of the present invention.

A conventional apparatus can include a fed-batch stirred reactor, abatch stirred reactor, a continuous flow stirred reactor withultrafiltration, and/or a continuous plug-flow column reactor (Fernandade Castilhos Corazza, Flávio Faria de Moraes, Gisella Maria Zanin andIvo Neitzel, 2003, Optimal control in fed-batch reactor for thecellobiose hydrolysis, Acta Scientiarum. Technology 25: 33-38; Gusakov,A. V., and Sinitsyn, A. P., 1985, Kinetics of the enzymatic hydrolysisof cellulose: 1. A mathematical model for a batch reactor process, Enz.Microb. Technol. 7: 346-352), an attrition reactor (Ryu, S. K., and Lee,J. M., 1983, Bioconversion of waste cellulose by using an attritionbioreactor, Biotechnol. Bioeng. 25: 53-65), or a reactor with intensivestirring induced by an electromagnetic field (Gusakov, A. V., Sinitsyn,A. P., Davydkin, I. Y Davydkin, V. Y., Protas, O. V., 1996, Enhancementof enzymatic cellulose hydrolysis using a novel type of bioreactor withintensive stirring induced by electromagnetic field, Appl. Biochem.Biotechnol. 56: 141-153). Additional reactor types include: fluidizedbed, upflow blanket, immobilized, and extruder type reactors forhydrolysis and/or fermentation.

Pretreatment.

In practicing the methods of the present invention, any pretreatmentprocess known in the art can be used to disrupt plant cell wallcomponents of the cellulosic material (Chandra et al., 2007, Substratepretreatment: The key to effective enzymatic hydrolysis oflignocellulosics? Adv. Biochem. Engin./Biotechnol. 108: 67-93; Galbe andZacchi, 2007, Pretreatment of lignocellulosic materials for efficientbioethanol production, Adv. Biochem. Engin./Biotechnol. 108: 41-65;Hendriks and Zeeman, 2009, Pretreatments to enhance the digestibility oflignocellulosic biomass, Bioresource Technol. 100: 10-18; Mosier et al.,2005, Features of promising technologies for pretreatment oflignocellulosic biomass, Bioresource Technol. 96: 673-686; Taherzadehand Karimi, 2008, Pretreatment of lignocellulosic wastes to improveethanol and biogas production: A review, Int. J. of Mol. Sci. 9:1621-1651; Yang and Wyman, 2008, Pretreatment: the key to unlockinglow-cost cellulosic ethanol, Biofuels Bioproducts andBiorefining-Biofpr. 2: 26-40).

The cellulosic material can also be subjected to particle sizereduction, pre-soaking, wetting, washing, and/or conditioning prior topretreatment using methods known in the art.

Conventional pretreatments include, but are not limited to, steampretreatment (with or without explosion), dilute acid pretreatment, hotwater pretreatment, alkaline pretreatment, lime pretreatment, wetoxidation, wet explosion, ammonia fiber explosion, organosolvpretreatment, and biological pretreatment. Additional pretreatmentsinclude ammonia percolation, ultrasound, electroporation, microwave,supercritical CO₂, supercritical H₂O, ozone, and gamma irradiationpretreatments.

The cellulosic material can be pretreated before hydrolysis and/orfermentation. Pretreatment is preferably performed prior to thehydrolysis. Alternatively, the pretreatment can be carried outsimultaneously with enzyme hydrolysis to release fermentable sugars,such as glucose, xylose, and/or cellobiose. In most cases thepretreatment step itself results in some conversion of the cellulosicmaterial to fermentable sugars (even in absence of enzymes).

Steam Pretreatment: In steam pretreatment, cellulosic material is heatedto disrupt the plant cell wall components, including lignin,hemicellulose, and cellulose to make the cellulose and other fractions,e.g., hemicellulose, accessible to enzymes. Cellulosic material ispassed to or through a reaction vessel where steam is injected toincrease the temperature to the required temperature and pressure and isretained therein for the desired reaction time. Steam pretreatment ispreferably done at 140-230° C., more preferably 160-200° C., and mostpreferably 170-190° C., where the optimal temperature range depends onany addition of a chemical catalyst. Residence time for the steampretreatment is preferably 1-15 minutes, more preferably 3-12 minutes,and most preferably 4-10 minutes, where the optimal residence timedepends on temperature range and any addition of a chemical catalyst.Steam pretreatment allows for relatively high solids loadings, so thatcellulosic material is generally only moist during the pretreatment. Thesteam pretreatment is often combined with an explosive discharge of thematerial after the pretreatment, which is known as steam explosion, thatis, rapid flashing to atmospheric pressure and turbulent flow of thematerial to increase the accessible surface area by fragmentation (Duffand Murray, 1996, Bioresource Technology 855: 1-33; Galbe and Zacchi,2002, Appl. Microbiol. Biotechnol. 59: 618-628; U.S. Patent ApplicationNo. 20020164730). During steam pretreatment, hemicellulose acetyl groupsare cleaved and the resulting acid autocatalyzes partial hydrolysis ofthe hemicellulose to monosaccharides and oligosaccharides. Lignin isremoved to only a limited extent.

A catalyst such as H₂SO₄ or SO₂ (typically 0.3 to 3% w/w) is often addedprior to steam pretreatment, which decreases the time and temperature,increases the recovery, and improves enzymatic hydrolysis (Ballesteroset al., 2006, Appl. Biochem. Biotechnol. 129-132: 496-508; Varga et al.,2004, Appl. Biochem. Biotechnol. 113-116: 509-523; Sassner et al., 2006,Enzyme Microb. Technol. 39: 756-762).

Chemical Pretreatment: The term “chemical treatment” refers to anychemical pretreatment that promotes the separation and/or release ofcellulose, hemicellulose, and/or lignin. Examples of suitable chemicalpretreatment processes include, for example, dilute acid pretreatment,lime pretreatment, wet oxidation, ammonia fiber/freeze explosion (AFEX),ammonia percolation (APR), and organosolv pretreatments.

In dilute acid pretreatment, cellulosic material is mixed with diluteacid, typically H₂SO₄, and water to form a slurry, heated by steam tothe desired temperature, and after a residence time flashed toatmospheric pressure. The dilute acid pretreatment can be performed witha number of reactor designs, e.g., plug-flow reactors, counter-currentreactors, or continuous counter-current shrinking bed reactors (Duff andMurray, 1996, supra; Schell et al., 2004, Bioresource Technol. 91:179-188; Lee et al., 1999, Adv. Biochem. Eng. Biotechnol. 65: 93-115).

Several methods of pretreatment under alkaline conditions can also beused. These alkaline pretreatments include, but are not limited to, limepretreatment, wet oxidation, ammonia percolation (APR), and ammoniafiber/freeze explosion (AFEX).

Lime pretreatment is performed with calcium carbonate, sodium hydroxide,or ammonia at low temperatures of 85-150° C. and residence times from 1hour to several days (Wyman et al., 2005, Bioresource Technol. 96:1959-1966; Mosier et al., 2005, Bioresource Technol. 96: 673-686). WO2006/110891, WO 2006/11899, WO 2006/11900, and WO 2006/110901 disclosepretreatment methods using ammonia.

Wet oxidation is a thermal pretreatment performed typically at 180-200°C. for 5-15 minutes with addition of an oxidative agent such as hydrogenperoxide or over-pressure of oxygen (Schmidt and Thomsen, 1998,Bioresource Technol. 64: 139-151; Palonen et al., 2004, Appl. Biochem.Biotechnol. 117: 1-17; Varga et al., 2004, Biotechnol. Bioeng. 88:567-574; Martin et al., 2006, J. Chem. Technol. Biotechnol. 81:1669-1677). The pretreatment is performed at preferably 1-40% drymatter, more preferably 2-30% dry matter, and most preferably 5-20% drymatter, and often the initial pH is increased by the addition of alkalisuch as sodium carbonate.

A modification of the wet oxidation pretreatment method, known as wetexplosion (combination of wet oxidation and steam explosion), can handledry matter up to 30%. In wet explosion, the oxidizing agent isintroduced during pretreatment after a certain residence time. Thepretreatment is then ended by flashing to atmospheric pressure (WO2006/032282).

Ammonia fiber explosion (AFEX) involves treating cellulosic materialwith liquid or gaseous ammonia at moderate temperatures such as 90-100°C. and high pressure such as 17-20 bar for 5-10 minutes, where the drymatter content can be as high as 60% (Gollapalli et al., 2002, Appl.Biochem. Biotechnol. 98: 23-35; Chundawat et al., 2007, Biotechnol.Bioeng. 96: 219-231; Alizadeh et al., 2005, Appl. Biochem. Biotechnol.121: 1133-1141; Teymouri et al., 2005, Bioresource Technol. 96:2014-2018). AFEX pretreatment results in the depolymerization ofcellulose and partial hydrolysis of hemicellulose. Lignin-carbohydratecomplexes are cleaved.

Organosolv pretreatment delignifies cellulosic material by extractionusing aqueous ethanol (40-60% ethanol) at 160-200° C. for 30-60 minutes(Pan et al., 2005, Biotechnol. Bioeng. 90: 473-481; Pan et al., 2006,Biotechnol. Bioeng. 94: 851-861; Kurabi et al., 2005, Appl. Biochem.Biotechnol. 121: 219-230). Sulphuric acid is usually added as acatalyst. In organosolv pretreatment, the majority of hemicellulose isremoved.

Other examples of suitable pretreatment methods are described by Schellet al., 2003, Appl. Biochem. and Biotechnol. Vol. 105-108, p. 69-85, andMosier et al., 2005, Bioresource Technology 96: 673-686, and U.S.Published Application 2002/0164730.

In one aspect, the chemical pretreatment is preferably carried out as anacid treatment, and more preferably as a continuous dilute and/or mildacid treatment. The acid is typically sulfuric acid, but other acids canalso be used, such as acetic acid, citric acid, nitric acid, phosphoricacid, tartaric acid, succinic acid, hydrogen chloride, or mixturesthereof. Mild acid treatment is conducted in the pH range of preferably1-5, more preferably 1-4, and most preferably 1-3. In one aspect, theacid concentration is in the range from preferably 0.01 to 20 wt % acid,more preferably 0.05 to 10 wt % acid, even more preferably 0.1 to 5 wt %acid, and most preferably 0.2 to 2.0 wt % acid. The acid is contactedwith cellulosic material and held at a temperature in the range ofpreferably 160-220° C., and more preferably 165-195° C., for periodsranging from seconds to minutes to, e.g., 1 second to 60 minutes.

In another aspect, pretreatment is carried out as an ammonia fiberexplosion step (AFEX pretreatment step).

In another aspect, pretreatment takes place in an aqueous slurry. Inpreferred aspects, cellulosic material is present during pretreatment inamounts preferably between 10-80 wt %, more preferably between 20-70 wt%, and most preferably between 30-60 wt %, such as around 50 wt %. Thepretreated cellulosic material can be unwashed or washed using anymethod known in the art, e.g., washed with water.

Mechanical Pretreatment: The term “mechanical pretreatment” refers tovarious types of grinding or milling (e.g., dry milling, wet milling, orvibratory ball milling).

Physical Pretreatment: The term “physical pretreatment” refers to anypretreatment that promotes the separation and/or release of cellulose,hemicellulose, and/or lignin from the cellulosic material. For example,physical pretreatment can involve irradiation (e.g., microwaveirradiation), steaming/steam explosion, hydrothermolysis, andcombinations thereof.

Physical pretreatment can involve high pressure and/or high temperature(steam explosion). In one aspect, high pressure means pressure in therange of preferably about 300 to about 600 psi, more preferably about350 to about 550 psi, and most preferably about 400 to about 500 psi,such as around 450 psi. In another aspect, high temperature meanstemperatures in the range of about 100 to about 300° C., preferablyabout 140 to about 235° C. In a preferred aspect, mechanicalpretreatment is performed in a batch-process, steam gun hydrolyzersystem that uses high pressure and high temperature as defined above,e.g., a Sunds Hydrolyzer available from Sunds Defibrator AB, Sweden.

Combined Physical and Chemical Pretreatment: Cellulosic material can bepretreated both physically and chemically. For instance, thepretreatment step can involve dilute or mild acid treatment and hightemperature and/or pressure treatment. The physical and chemicalpretreatments can be carried out sequentially or simultaneously, asdesired. A mechanical pretreatment can also be included.

Accordingly, in a preferred aspect, the cellulosic material is subjectedto mechanical, chemical, or physical pretreatment, or any combinationthereof, to promote the separation and/or release of cellulose,hemicellulose, and/or lignin.

Biological Pretreatment: The term “biological pretreatment” refers toany biological pretreatment that promotes the separation and/or releaseof cellulose, hemicellulose, and/or lignin from the cellulosic material.Biological pretreatment techniques can involve applyinglignin-solubilizing microorganisms (see, for example, Hsu, T.-A., 1996,Pretreatment of biomass, in Handbook on Bioethanol: Production andUtilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C.,179-212; Ghosh and Singh, 1993, Physicochemical and biologicaltreatments for enzymatic/microbial conversion of cellulosic biomass,Adv. Appl. Microbiol. 39: 295-333; McMillan, J. D., 1994, Pretreatinglignocellulosic biomass: a review, in Enzymatic Conversion of Biomassfor Fuels Production, Himmel, M. E., Baker, J. O., and Overend, R. P.,eds., ACS Symposium Series 566, American Chemical Society, Washington,D.C., chapter 15; Gong, C. S., Cao, N. J., Du, J., and Tsao, G. T.,1999, Ethanol production from renewable resources, in Advances inBiochemical Engineering/Biotechnology, Scheper, T., ed., Springer-VerlagBerlin Heidelberg, Germany, 65: 207-241; Olsson and Hahn-Hagerdal, 1996,Fermentation of lignocellulosic hydrolysates for ethanol production,Enz. Microb. Tech. 18: 312-331; and Vallander and Eriksson, 1990,Production of ethanol from lignocellulosic materials: State of the art,Adv. Biochem. Eng./Biotechnol. 42: 63-95).

Saccharification.

In the hydrolysis step, also known as saccharification, the cellulosicmaterial, e.g., pretreated, is hydrolyzed to break down cellulose andalternatively also hemicellulose to fermentable sugars, such as glucose,cellobiose, xylose, xylulose, arabinose, mannose, galactose, and/orsoluble oligosaccharides. The hydrolysis is performed enzymatically byan enzyme composition in the presence of a variant havingcellobiohydrolase II activity. The enzyme and protein components of thecompositions can be added sequentially.

Enzymatic hydrolysis is preferably carried out in a suitable aqueousenvironment under conditions that can be readily determined by oneskilled in the art. In a preferred aspect, hydrolysis is performed underconditions suitable for the activity of the enzyme(s), i.e., optimal forthe enzyme(s). The hydrolysis can be carried out as a fed batch orcontinuous process where the pretreated cellulosic material (substrate)is fed gradually to, for example, an enzyme containing hydrolysissolution.

The saccharification is generally performed in stirred-tank reactors orfermentors under controlled pH, temperature, and mixing conditions.Suitable process time, temperature and pH conditions can readily bedetermined by one skilled in the art. For example, the saccharificationcan last up to 200 hours, but is typically performed for preferablyabout 12 to about 96 hours, more preferably about 16 to about 72 hours,and most preferably about 24 to about 48 hours. The temperature is inthe range of preferably about 25° C. to about 70° C., more preferablyabout 30° C. to about 65° C., and more preferably about 40° C. to 60°C., in particular about 50° C. The pH is in the range of preferablyabout 3 to about 8, more preferably about 3.5 to about 7, and mostpreferably about 4 to about 6, in particular about pH 5. The dry solidscontent is in the range of preferably about 5 to about 50 wt %, morepreferably about 10 to about 40 wt %, and most preferably about 20 toabout 30 wt %.

The optimum amounts of the enzymes and variants having cellobiohydrolaseII activity depend on several factors including, but not limited to, themixture of component cellulolytic enzymes, the cellulosic substrate, theconcentration of cellulosic substrate, the pretreatment(s) of thecellulosic substrate, temperature, time, pH, and inclusion of fermentingorganism (e.g., yeast for Simultaneous Saccharification andFermentation).

In one aspect, an effective amount of cellulolytic or hemicellulolyticenzyme protein to cellulosic material is about 0.5 to about 50 mg,preferably at about 0.5 to about 40 mg, more preferably at about 0.5 toabout 25 mg, more preferably at about 0.75 to about 20 mg, morepreferably at about 0.75 to about 15 mg, even more preferably at about0.5 to about 10 mg, and most preferably at about 2.5 to about 10 mg perg of cellulosic material.

In another aspect, an effective amount of a variant havingcellobiohydrolase II activity to cellulosic material is about 0.01 toabout 50.0 mg, preferably about 0.01 to about 40 mg, more preferablyabout 0.01 to about 30 mg, more preferably about 0.01 to about 20 mg,more preferably about 0.01 to about 10 mg, more preferably about 0.01 toabout 5 mg, more preferably at about 0.025 to about 1.5 mg, morepreferably at about 0.05 to about 1.25 mg, more preferably at about0.075 to about 1.25 mg, more preferably at about 0.1 to about 1.25 mg,even more preferably at about 0.15 to about 1.25 mg, and most preferablyat about 0.25 to about 1.0 mg per g of cellulosic material.

In another aspect, an effective amount of a variant havingcellobiohydrolase II activity to cellulolytic enzyme protein is about0.005 to about 1.0 g, preferably at about 0.01 to about 1.0 g, morepreferably at about 0.15 to about 0.75 g, more preferably at about 0.15to about 0.5 g, more preferably at about 0.1 to about 0.5 g, even morepreferably at about 0.1 to about 0.5 g, and most preferably at about0.05 to about 0.2 g per g of cellulolytic enzyme protein.

The enzyme compositions can comprise any protein that is useful indegrading or converting a cellulosic material.

In one aspect, the enzyme composition comprises or further comprises oneor more (several) proteins selected from the group consisting of acellulase, a GH61 polypeptide having cellulolytic enhancing activity, ahemicellulase, an expansin, an esterase, a laccase, a ligninolyticenzyme, a pectinase, a peroxidase, a protease, and a swollenin. Inanother aspect, the cellulase is preferably one or more (several)enzymes selected from the group consisting of an endoglucanase, acellobiohydrolase, and a beta-glucosidase. In another aspect, thehemicellulase is preferably one or more (several) enzymes selected fromthe group consisting of an acetylmannan esterase, an acetyxylanesterase, an arabinanase, an arabinofuranosidase, a coumaric acidesterase, a feruloyl esterase, a galactosidase, a glucuronidase, aglucuronoyl esterase, a mannanase, a mannosidase, a xylanase, and axylosidase.

In another aspect, the enzyme composition comprises one or more(several) cellulolytic enzymes. In another aspect, the enzymecomposition comprises or further comprises one or more (several)hemicellulolytic enzymes. In another aspect, the enzyme compositioncomprises one or more (several) cellulolytic enzymes and one or more(several) hemicellulolytic enzymes. In another aspect, the enzymecomposition comprises one or more (several) enzymes selected from thegroup of cellulolytic enzymes and hemicellulolytic enzymes. In anotheraspect, the enzyme composition comprises an endoglucanase. In anotheraspect, the enzyme composition comprises a cellobiohydrolase. In anotheraspect, the enzyme composition comprises a beta-glucosidase. In anotheraspect, the enzyme composition comprises a polypeptide havingcellulolytic enhancing activity. In another aspect, the enzymecomposition comprises an endoglucanase and a polypeptide havingcellulolytic enhancing activity. In another aspect, the enzymecomposition comprises a cellobiohydrolase and a polypeptide havingcellulolytic enhancing activity. In another aspect, the enzymecomposition comprises a beta-glucosidase and a polypeptide havingcellulolytic enhancing activity. In another aspect, the enzymecomposition comprises an endoglucanase and a cellobiohydrolase. Inanother aspect, the enzyme composition comprises an endoglucanase and abeta-glucosidase. In another aspect, the enzyme composition comprises acellobiohydrolase and a beta-glucosidase. In another aspect, the enzymecomposition comprises an endoglucanase, a cellobiohydrolase, and apolypeptide having cellulolytic enhancing activity. In another aspect,the enzyme composition comprises an endoglucanase, a beta-glucosidase,and a polypeptide having cellulolytic enhancing activity. In anotheraspect, the enzyme composition comprises a cellobiohydrolase, abeta-glucosidase, and a polypeptide having cellulolytic enhancingactivity. In another aspect, the enzyme composition comprises anendoglucanase, a cellobiohydrolase, and a beta-glucosidase, and apolypeptide having cellulolytic enhancing activity.

In another aspect, the enzyme composition comprises an acetylmannanesterase. In another aspect, the enzyme composition comprises anacetyxylan esterase. In another aspect, the enzyme composition comprisesan arabinanase (e.g., alpha-L-arabinanase). In another aspect, theenzyme composition comprises an arabinofuranosidase (e.g.,alpha-L-arabinofuranosidase). In another aspect, the enzyme compositioncomprises a coumaric acid esterase. In another aspect, the enzymecomposition comprises a feruloyl esterase. In another aspect, the enzymecomposition comprises a galactosidase (e.g., alpha-galactosidase and/orbeta-galactosidase). In another aspect, the enzyme composition comprisesa glucuronidase (e.g., alpha-D-glucuronidase). In another aspect, theenzyme composition comprises a glucuronoyl esterase. In another aspect,the enzyme composition comprises a mannanase. In another aspect, theenzyme composition comprises a mannosidase (e.g., beta-mannosidase). Inanother aspect, the enzyme composition comprises a xylanase. In apreferred aspect, the xylanase is a Family 10 xylanase. In anotheraspect, the enzyme composition comprises a xylosidase. In anotheraspect, the enzyme composition comprises an expansin. In another aspect,the enzyme composition comprises an esterase. In another aspect, theenzyme composition comprises a laccase. In another aspect, the enzymecomposition comprises a ligninolytic enzyme. In a preferred aspect, theligninolytic enzyme is a manganese peroxidase. In another preferredaspect, the ligninolytic enzyme is a lignin peroxidase. In anotherpreferred aspect, the ligninolytic enzyme is a H₂O₂-producing enzyme. Inanother aspect, the enzyme composition comprises a pectinase. In anotheraspect, the enzyme composition comprises a peroxidase. In anotheraspect, the enzyme composition comprises a protease. In another aspect,the enzyme composition comprises a swollenin.

In the methods of the present invention, the enzyme(s) can be addedprior to or during fermentation, e.g., during saccharification or duringor after propagation of the fermenting microorganism(s).

One or more (several) components of the enzyme composition may bewild-type proteins, recombinant proteins, or a combination of wild-typeproteins and recombinant proteins. For example, one or more (several)components may be native proteins of a cell, which is used as a hostcell to express recombinantly one or more (several) other components ofthe enzyme composition. One or more (several) components of the enzymecomposition may be produced as monocomponents, which are then combinedto form the enzyme composition. The enzyme composition may be acombination of multicomponent and monocomponent protein preparations.

The enzymes used in the methods of the present invention may be in anyform suitable for use, such as, for example, a crude fermentation brothwith or without cells removed, a cell lysate with or without cellulardebris, a semi-purified or purified enzyme preparation, or a host cellas a source of the enzymes. The enzyme composition may be a dry powderor granulate, a non-dusting granulate, a liquid, a stabilized liquid, ora stabilized protected enzyme. Liquid enzyme preparations may, forinstance, be stabilized by adding stabilizers such as a sugar, a sugaralcohol or another polyol, and/or lactic acid or another organic acidaccording to established processes.

The enzymes can be derived or obtained from any suitable origin,including, bacterial, fungal, yeast, plant, or mammalian origin. Theterm “obtained” means herein that the enzyme may have been isolated froman organism that naturally produces the enzyme as a native enzyme. Theterm “obtained” also means herein that the enzyme may have been producedrecombinantly in a host organism employing methods described herein,wherein the recombinantly produced enzyme is either native or foreign tothe host organism or has a modified amino acid sequence, e.g., havingone or more (several) amino acids that are deleted, inserted and/orsubstituted, i.e., a recombinantly produced enzyme that is a mutantand/or a fragment of a native amino acid sequence or an enzyme producedby nucleic acid shuffling processes known in the art. Encompassed withinthe meaning of a native enzyme are natural variants and within themeaning of a foreign enzyme are variants obtained recombinantly, such asby site-directed mutagenesis or shuffling.

The polypeptide having enzyme activity may be a bacterial polypeptide.For example, the polypeptide may be a gram positive bacterialpolypeptide such as a Bacillus, Streptococcus, Streptomyces,Staphylococcus, Enterococcus, Lactobacillus, Lactococcus, Clostridium,Geobacillus, or Oceanobacillus polypeptide having enzyme activity, or aGram negative bacterial polypeptide such as an E. coli, Pseudomonas,Salmonella, Campylobacter, Helicobacter, Flavobacterium, Fusobacterium,Ilyobacter, Neisseria, or Ureaplasma polypeptide having enzyme activity.

In a preferred aspect, the polypeptide is a Bacillus alkalophilus,Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans,Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus,Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacilluspumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis polypeptide having enzyme activity.

In another preferred aspect, the polypeptide is a Streptococcusequisimilis, Streptococcus pyogenes, Streptococcus uberis, orStreptococcus equi subsp. Zooepidemicus polypeptide having enzymeactivity.

In another preferred aspect, the polypeptide is a Streptomycesachromogenes, Streptomyces avermitilis, Streptomyces coelicolor,Streptomyces griseus, or Streptomyces lividans polypeptide having enzymeactivity.

The polypeptide having enzyme activity may also be a fungal polypeptide,and more preferably a yeast polypeptide such as a Candida,Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowiapolypeptide having enzyme activity; or more preferably a filamentousfungal polypeptide such as an Acremonium, Agaricus, Alternaria,Aspergillus, Aureobasidium, Botryosphaeria, Ceriporiopsis, Chaetomidium,Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes,Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium,Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula,Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor,Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium,Phanerochaete, Piromyces, Poitrasia, Pseudoplectania,Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces,Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea,Verticillium, Volvariella, or Xylaria polypeptide having enzymeactivity.

In a preferred aspect, the polypeptide is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomycesnorbensis, or Saccharomyces oviformis polypeptide having enzymeactivity.

In another preferred aspect, the polypeptide is an Acremoniumcellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillusfumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillusnidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporiumkeratinophilum, Chrysosporium lucknowense, Chrysosporium tropicum,Chrysosporium merdarium, Chrysosporium inops, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium zonatum, Fusariumbactridioides, Fusarium cerealis, Fusarium crookwellense, Fusariumculmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sporotrichioides, Fusarium suiphureum, Fusarium torulosum,Fusarium trichothecioides, Fusarium venenatum, Humicola grisea, Humicolainsolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum,Penicillium purpurogenum, Phanerochaete chrysosporium, Thielaviaachromatica, Thielavia albomyces, Thielavia albopilosa, Thielaviaaustraleinsis, Thielavia fimeti, Thielavia microspora, Thielaviaovispora, Thielavia peruviana, Thielavia spededonium, Thielavia setosa,Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei,Trichoderma viride, or Trichophaea saccata polypeptide having enzymeactivity.

Chemically modified or protein engineered mutants of the polypeptideshaving enzyme activity may also be used.

One or more (several) components of the enzyme composition may be arecombinant component, i.e., produced by cloning of a DNA sequenceencoding the single component and subsequent cell transformed with theDNA sequence and expressed in a host (see, for example, WO 91/17243 andWO 91/17244). The host is preferably a heterologous host (enzyme isforeign to host), but the host may under certain conditions also be ahomologous host (enzyme is native to host). Monocomponent cellulolyticenzymes may also be prepared by purifying such a protein from afermentation broth.

In one aspect, the one or more (several) cellulolytic enzymes comprise acommercial cellulolytic enzyme preparation. Examples of commercialcellulolytic enzyme preparations suitable for use in the presentinvention include, for example, CELLIC™ CTec (Novozymes A/S),CELLUCLAST™ (Novozymes A/S), NOVOZYM™ 188 (Novozymes A/S), CELLUZYME™(Novozymes A/S), CEREFLO™ (Novozymes A/S), and ULTRAFLO™ (NovozymesA/S), ACCELERASE™ (Genencor Int.), LAMINEX™ (Genencor Int.), SPEZYME™ CP(Genencor Int.), ROHAMENT™ 7069 W (Röhm GmbH), FIBREZYME® LDI (DyadicInternational, Inc.), FIBREZYME® LBR (Dyadic International, Inc.), orVISCOSTAR® 150L (Dyadic International, Inc.). The cellulase enzymes areadded in amounts effective from about 0.001 to about 5.0 wt % of solids,more preferably from about 0.025 to about 4.0 wt % of solids, and mostpreferably from about 0.005 to about 2.0 wt % of solids.

Examples of bacterial endoglucanases that can be used in the methods ofthe present invention, include, but are not limited to, an Acidothermuscellulolyticus endoglucanase (WO 91/05039; WO 93/15186; U.S. Pat. No.5,275,944; WO 96/02551; U.S. Pat. No. 5,536,655, WO 00/70031, WO05/093050); Thermobifida fusca endoglucanase III (WO 05/093050); andThermobifida fusca endoglucanase V (WO 05/093050).

Examples of fungal endoglucanases that can be used in the presentinvention include, but are not limited to, a Trichoderma reeseiendoglucanase I (Penttila et al., 1986, Gene 45: 253-263; Trichodermareesei Cel7B endoglucanase I; GENBANK™ accession no. M15665; SEQ ID NO:4); Trichoderma reesei endoglucanase II (Saloheimo, et al., 1988, Gene63:11-22; Trichoderma reesei Cel5A endoglucanase II; GENBANK™ accessionno. M19373; SEQ ID NO: 6); Trichoderma reesei endoglucanase III (Okadaet al., 1988, Appl. Environ. Microbiol. 64: 555-563; GENBANK™ accessionno. AB003694; SEQ ID NO: 8); Trichoderma reesei endoglucanase V(Saloheimo et al., 1994, Molecular Microbiology 13: 219-228; GENBANK™accession no. Z33381; SEQ ID NO: 10); Aspergillus aculeatusendoglucanase (Ooi et al., 1990, Nucleic Acids Research 18: 5884);Aspergillus kawachii endoglucanase (Sakamoto et al., 1995, CurrentGenetics 27: 435-439); Erwinia carotovara endoglucanase (Saarilahti etal., 1990, Gene 90: 9-14); Fusarium oxysporum endoglucanase (GENBANK™accession no. L29381); Humicola grisea var. thermoidea endoglucanase(GENBANK™ accession no. AB003107); Melanocarpus albomyces endoglucanase(GENBANK™ accession no. MAL515703); Neurospora crassa endoglucanase(GENBANK™ accession no. XM_324477); Humicola insolens endoglucanase V(SEQ ID NO: 12); Myceliophthora thermophila CBS 117.65 endoglucanase(SEQ ID NO: 14); basidiomycete CBS 495.95 endoglucanase (SEQ ID NO: 16);basidiomycete CBS 494.95 endoglucanase (SEQ ID NO: 18); Thielaviaterrestris NRRL 8126 CEL6B endoglucanase (SEQ ID NO: 20); Thielaviaterrestris NRRL 8126 CEL6C endoglucanase (SEQ ID NO: 22); Thielaviaterrestris NRRL 8126 CEL7C endoglucanase (SEQ ID NO: 24); Thielaviaterrestris NRRL 8126 CEL7E endoglucanase (SEQ ID NO: 26); Thielaviaterrestris NRRL 8126 CEL7F endoglucanase (SEQ ID NO: 28); Cladorrhinumfoecundissimum ATCC 62373 CEL7A endoglucanase (SEQ ID NO: 30); andTrichoderma reesei strain No. VTT-D-80133 endoglucanase (SEQ ID NO: 32;GENBANK™ accession no. M15665). The endoglucanases of SEQ ID NO: 4, SEQID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24,SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, and SEQ ID NO: 32,described above are encoded by the mature polypeptide coding sequence ofSEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11,SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, and SEQID NO: 31, respectively.

Examples of cellobiohydrolases useful in the present invention include,but are not limited to, Trichoderma reesei cellobiohydrolase I (SEQ IDNO: 34); Trichoderma reesei cellobiohydrolase II (SEQ ID NO: 36);Humicola insolens cellobiohydrolase I (SEQ ID NO: 38); Myceliophthorathermophila cellobiohydrolase II (SEQ ID NO: 40 and SEQ ID NO: 42);Thielavia terrestris cellobiohydrolase II (CEL6A) (SEQ ID NO: 2);Chaetomium thermophilum cellobiohydrolase I (SEQ ID NO: 44); andChaetomium thermophilum cellobiohydrolase II (SEQ ID NO: 46),Aspergillus fumigatus cellobiohydrolase I (SEQ ID NO: 48), andAspergillus fumigatus cellobiohydrolase II (SEQ ID NO: 50). Thecellobiohydrolases of SEQ ID NO: 2, SEQ ID NO: 34, SEQ ID NO: 36, SEQ IDNO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQID NO: 48, and SEQ ID NO: 50, described above are encoded by the maturepolypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 33, SEQ ID NO:35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ IDNO: 45, SEQ ID NO: 47, and SEQ ID NO: 49, respectively.

Examples of beta-glucosidases useful in the present invention include,but are not limited to, Aspergillus oryzae beta-glucosidase (SEQ ID NO:52); Aspergillus fumigatus beta-glucosidase (SEQ ID NO: 54); Penicilliumbrasilianum IBT 20888 beta-glucosidase (SEQ ID NO: 56); Aspergillusnigerbeta-glucosidase (SEQ ID NO: 58); and Aspergillus aculeatusbeta-glucosidase (SEQ ID NO: 60). The beta-glucosidases of SEQ ID NO:52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, and SEQ ID NO: 60,described above are encoded by the mature polypeptide coding sequence ofSEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, and SEQ IDNO: 59, respectively.

Examples of other beta-glucosidases useful in the present inventioninclude a Aspergillus oryzae beta-glucosidase variant fusion protein ofSEQ ID NO: 62 or the Aspergillus oryzae beta-glucosidase fusion proteinof SEQ ID NO: 64. The beta-glucosidase fusion proteins of SEQ ID NO: 62and SEQ ID NO: 64 are encoded by SEQ ID NO: 61 and SEQ ID NO: 63,respectively.

The Aspergillus oryzae beta-glucosidase can be obtained according to WO2002/095014. The Aspergillus fumigatus beta-glucosidase can be obtainedaccording to WO 2005/047499. The Penicillium brasilianumbeta-glucosidase can be obtained according to WO 2007/019442. TheAspergillus niger beta-glucosidase can be obtained according to Dan etal., 2000, J. Biol. Chem. 275: 4973-4980. The Aspergillus aculeatusbeta-glucosidase can be obtained according to Kawaguchi et al., 1996,Gene 173: 287-288.

Other useful endoglucanases, cellobiohydrolases, and beta-glucosidasesare disclosed in numerous Glycosyl Hydrolase families using theclassification according to Henrissat B., 1991, A classification ofglycosyl hydrolases based on amino-acid sequence similarities, Biochem.J. 280: 309-316, and Henrissat B., and Bairoch A., 1996, Updating thesequence-based classification of glycosyl hydrolases, Biochem. J. 316:695-696.

Other cellulolytic enzymes that may be useful in the present inventionare described in EP 495,257, EP 531,315, EP 531,372, WO 89/09259, WO94/07998, WO 95/24471, WO 96/11262, WO 96/29397, WO 96/034108, WO97/14804, WO 98/08940, WO 98/012307, WO 98/13465, WO 98/015619, WO98/015633, WO 98/028411, WO 99/06574, WO 99/10481, WO 99/025846, WO99/025847, WO 99/031255, WO 2000/009707, WO 2002/050245, WO2002/0076792, WO 2002/101078, WO 2003/027306, WO 2003/052054, WO2003/052055, WO 2003/052056, WO 2003/052057, WO 2003/052118, WO2004/016760, WO 2004/043980, WO 2004/048592, WO 2005/001065, WO2005/028636, WO 2005/093050, WO 2005/093073, WO 2006/074005, WO2006/117432, WO 2007/071818, WO 2007/071820, WO 2008/008070, WO2008/008793, U.S. Pat. No. 4,435,307, U.S. Pat. No. 5,457,046, U.S. Pat.No. 5,648,263, U.S. Pat. No. 5,686,593, U.S. Pat. No. 5,691,178, U.S.Pat. No. 5,763,254, and U.S. Pat. No. 5,776,757.

In the methods of the present invention, any GH61 polypeptide havingcellulolytic enhancing activity can be used.

In a first aspect, the polypeptide having cellulolytic enhancingactivity comprises the following motifs:

[ILMV]-P-X(4,5)-G-X-Y-[ILMV]-X—R-X-[EQ]-X(4)-[HNQ] and[FW]-[TF]-K-[AIV],

wherein X is any amino acid, X(4,5) is any amino acid at 4 or 5contiguous positions, and X(4) is any amino acid at 4 contiguouspositions.

The polypeptide comprising the above-noted motifs may further comprise:

H-X(1,2)-G-P-X(3)-[YW]-[AILMV],

[EQ]-X—Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV], or

H-X(1,2)-G-P-X(3)-[YW]-[AILMV] and[EQ]-X—Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV],

wherein X is any amino acid, X(1,2) is any amino acid at 1 position or 2contiguous positions, X(3) is any amino acid at 3 contiguous positions,and X(2) is any amino acid at 2 contiguous positions. In the abovemotifs, the accepted IUPAC single letter amino acid abbreviation isemployed.

In a preferred aspect, the polypeptide having cellulolytic enhancingactivity further comprises H-X(1,2)-G-P-X(3)-[YW]-[AILMV]. In anotherpreferred aspect, the isolated polypeptide having cellulolytic enhancingactivity further comprises [EQ]-X—Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV]. Inanother preferred aspect, the polypeptide having cellulolytic enhancingactivity further comprises H-X(1,2)-G-P-X(3)-[YW]-[AILMV] and[EQ]-X—Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV].

In a second aspect, the polypeptide having cellulolytic enhancingactivity comprises the following motif:

[ILMV]-P-x(4,5)-G-x-Y-[ILMV]-x-R-x-[EQ]-x(3)-A-[HNQ],

wherein x is any amino acid, x(4,5) is any amino acid at 4 or 5contiguous positions, and x(3) is any amino acid at 3 contiguouspositions. In the above motif, the accepted IUPAC single letter aminoacid abbreviation is employed.

In a third aspect, the polypeptide having cellulolytic enhancingactivity comprises an amino acid sequence that has a degree of identityto the mature polypeptide of SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO:70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ IDNO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98,SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ IDNO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116,SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ IDNO: 126, or SEQ ID NO: 128 of at least 60%, e.g., at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, or at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or at least 100%.

In a fourth aspect, the polypeptide having cellulolytic enhancingactivity is encoded by a polynucleotide that hybridizes under at leastvery low stringency conditions, preferably at least low stringencyconditions, more preferably at least medium stringency conditions, morepreferably at least medium-high stringency conditions, even morepreferably at least high stringency conditions, and most preferably atleast very high stringency conditions with (i) the mature polypeptidecoding sequence of SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ IDNO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89,SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO:99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO:117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, orSEQ ID NO: 127, (ii) the cDNA sequence contained in the maturepolypeptide coding sequence of SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO:75, or SEQ ID NO: 79, or the genomic DNA sequence comprising the maturepolypeptide coding sequence of SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO:69, SEQ ID NO: 77, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ IDNO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO:105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO:123, SEQ ID NO: 125, or SEQ ID NO: 127, (iii) a subsequence of (i) or(ii), or (iv) a full-length complementary strand of (i), (ii), or (iii)(J. Sambrook, E. F. Fritsch, and T. Maniatus, 1989, supra). Asubsequence of the mature polypeptide coding sequence of SEQ ID NO: 65,SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO:75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ IDNO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103,SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ IDNO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121,SEQ ID NO: 123, SEQ ID NO: 125, or SEQ ID NO: 127 contains at least 100contiguous nucleotides or preferably at least 200 contiguousnucleotides. Moreover, the subsequence may encode a polypeptide fragmentthat has cellulolytic enhancing activity.

In a fifth aspect, the polypeptide having cellulolytic enhancingactivity is encoded by a polynucleotide comprising or consisting of anucleotide sequence that has a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO:69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ IDNO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97,SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ IDNO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115,SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ IDNO: 125, or SEQ ID NO: 127 of preferably at least 60%, more preferablyat least 65%, more preferably at least 70%, more preferably at least75%, more preferably at least 80%, more preferably at least 85%, evenmore preferably at least 90%, most preferably at least 91%, at least92%, at least 93%, at least 94%, or at least 95%, and even mostpreferably at least 96%, at least 97%, at least 98%, at least 99%, or atleast 100%.

In a sixth aspect, the polypeptide having cellulolytic enhancingactivity is an artificial variant comprising a substitution, deletion,and/or insertion of one or more (or several) amino acids of the maturepolypeptide of SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO:72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ IDNO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100,SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ IDNO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118,SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, or SEQID NO: 128; or a homologous sequence thereof.

Preferably, amino acid changes are of a minor nature, that isconservative amino acid substitutions or insertions that do notsignificantly affect the folding and/or activity of the protein; smalldeletions, typically of one to about 30 amino acids; small amino- orcarboxyl-terminal extensions, such as an amino-terminal methionineresidue; a small linker peptide of up to about 20-25 residues; or asmall extension that facilitates purification by changing net charge oranother function, such as a poly-histidine tract, an antigenic epitopeor a binding domain.

Examples of conservative substitutions are within the group of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. The mostcommonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser,Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg,Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in a parent polypeptide can be identifiedaccording to procedures known in the art, such as site-directedmutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989,Science 244: 1081-1085). In the latter technique, single alaninemutations are introduced at every residue in the molecule, and theresultant mutant molecules are tested for cellulolytic enhancingactivity to identify amino acid residues that are critical to theactivity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem.271: 4699-4708. The active site of the enzyme or other biologicalinteraction can also be determined by physical analysis of structure, asdetermined by such techniques as nuclear magnetic resonance,crystallography, electron diffraction, or photoaffinity labeling, inconjunction with mutation of putative contact site amino acids. See, forexample, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992,J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64. The identities of essential amino acids can also be inferred fromanalysis of identities with polypeptides that are related to the parentpolypeptide.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

The total number of amino acid substitutions, deletions and/orinsertions of the mature polypeptide of SEQ ID NO: 66, SEQ ID NO: 68,SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO:78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ IDNO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO:106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO:124, SEQ ID NO: 126, or SEQ ID NO: 128 is not more than 4, e.g., 1, 2,3, or 4.

In one aspect, the one or more (several) hemicellulolytic enzymescomprise a commercial hemicellulolytic enzyme preparation. Examples ofcommercial hemicellulolytic enzyme preparations suitable for use in thepresent invention include, for example, SHEARZYME™ (Novozymes A/S),CELLIC™ HTec (Novozymes A/S), VISCOZYME® (Novozymes A/S), ULTRAFLO®(Novozymes A/S), PULPZYME® HC (Novozymes A/S), MULTIFECT® Xylanase(Genencor), ECOPULP® TX-200A (AB Enzymes), HSP 6000 Xylanase (DSM),DEPOL™ 333P (Biocatalysts Limit, Wales, UK), DEPOL™ 740L. (BiocatalystsLimit, Wales, UK), and DEPOL™ 762P (Biocatalysts Limit, Wales, UK).

Examples of xylanases useful in the methods of the present inventioninclude, but are not limited to, Aspergillus aculeatus xylanase(GeneSeqP:AAR63790; WO 94/21785), Aspergillus fumigatusxylanases (WO2006/078256; xyl 3 SEQ ID NO: 129 [DNA sequence] and SEQ ID NO: 130[deduced amino acid sequence]), and Thielavia terrestris NRRL 8126xylanases (WO 2009/079210).

Examples of beta-xylosidases useful in the methods of the presentinvention include, but are not limited to, Trichoderma reeseibeta-xylosidase (UniProtKB/TrEMBL accession number Q92458; SEQ ID NO:131 [DNA sequence] and SEQ ID NO: 132 [deduced amino acid sequence]),Talaromyces emersonii (SwissProt accession number Q8X212), andNeurospora crassa (SwissProt accession number Q7SOW4).

Examples of acetylxylan esterases useful in the methods of the presentinvention include, but are not limited to, Hypocrea jecorina acetylxylanesterase (WO 2005/001036), Neurospora crassa acetylxylan esterase(UniProt accession number q7s259), Thielavia terrestris NRRL 8126acetylxylan esterase (WO 2009/042846), Chaetomium globosum acetylxylanesterase (Uniprot accession number Q2GWX4), Chaetomium gracileacetylxylan esterase (GeneSeqP accession number AAB82124), Phaeosphaerianodorum acetylxylan esterase (Uniprot accession number Q0UHJ1), andHumicola insolens DSM 1800 acetylxylan esterase (WO 2009/073709).

Examples of ferulic acid esterases useful in the methods of the presentinvention include, but are not limited to, Humicola insolens DSM 1800feruloyl esterase (WO 2009/076122), Neurospora crassa feruloyl esterase(UniProt accession number Q9HGR3), and Neosartorya fischeri feruloylesterase (UniProt Accession number A1D9T4).

Examples of arabinofuranosidases useful in the methods of the presentinvention include, but are not limited to, Humicola insolens DSM 1800arabinofuranosidase (WO 2009/073383) and Aspergillus nigerarabinofuranosidase (GeneSeqP accession number AAR94170).

Examples of alpha-glucuronidases useful in the methods of the presentinvention include, but are not limited to, Aspergillus clavatusalpha-glucuronidase (UniProt accession number alccl 2), Trichodermareesei alpha-glucuronidase (Uniprot accession number Q99024),Talaromyces emersonii alpha-glucuronidase (UniProt accession numberQ8X211), Aspergillus niger alpha-glucuronidase (Uniprot accession numberQ96WX9), Aspergillus terreus alpha-glucuronidase (SwissProt accessionnumber Q0CJP9), and Aspergillus fumigatus alpha-glucuronidase (SwissProtaccession number Q4VWV45).

The enzymes and proteins used in the methods of the present inventionmay be produced by fermentation of the above-noted microbial strains ona nutrient medium containing suitable carbon and nitrogen sources andinorganic salts, using procedures known in the art (see, e.g., Bennett,J. W. and LaSure, L. (eds.), More Gene Manipulations in Fungi, AcademicPress, C A, 1991). Suitable media are available from commercialsuppliers or may be prepared according to published compositions (e.g.,in catalogues of the American Type Culture Collection). Temperatureranges and other conditions suitable for growth and enzyme productionare known in the art (see, e.g., Bailey, J. E., and Ollis, D. F.,Biochemical Engineering Fundamentals, McGraw-Hill Book Company, N Y,1986).

The fermentation can be any method of cultivation of a cell resulting inthe expression or isolation of an enzyme. Fermentation may, therefore,be understood as comprising shake flask cultivation, or small- orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing the enzymeto be expressed or isolated. The resulting enzymes produced by themethods described above may be recovered from the fermentation mediumand purified by conventional procedures.

Fermentation.

The fermentable sugars obtained from the hydrolyzed cellulosic materialcan be fermented by one or more (several) fermenting microorganismscapable of fermenting the sugars directly or indirectly into a desiredfermentation product. “Fermentation” or “fermentation process” refers toany fermentation process or any process comprising a fermentation step.Fermentation processes also include fermentation processes used in theconsumable alcohol industry (e.g., beer and wine), dairy industry (e.g.,fermented dairy products), leather industry, and tobacco industry. Thefermentation conditions depend on the desired fermentation product andfermenting organism and can easily be determined by one skilled in theart.

In the fermentation step, sugars, released from the cellulosic materialas a result of the pretreatment and enzymatic hydrolysis steps, arefermented to a product, e.g., ethanol, by a fermenting organism, such asyeast. Hydrolysis (saccharification) and fermentation can be separate orsimultaneous, as described herein.

Any suitable hydrolyzed cellulosic material can be used in thefermentation step in practicing the present invention. The material isgenerally selected based on the desired fermentation product, i.e., thesubstance to be obtained from the fermentation, and the processemployed, as is well known in the art.

The term “fermentation medium” is understood herein to refer to a mediumbefore the fermenting microorganism(s) is(are) added, such as, a mediumresulting from a saccharification process, as well as a medium used in asimultaneous saccharification and fermentation process (SSF).

“Fermenting microorganism” refers to any microorganism, includingbacterial and fungal organisms, suitable for use in a desiredfermentation process to produce a fermentation product. The fermentingorganism can be C₆ and/or C₅ fermenting organisms, or a combinationthereof. Both C₆ and C₅ fermenting organisms are well known in the art.Suitable fermenting microorganisms are able to ferment, i.e., convert,sugars, such as glucose, xylose, xylulose, arabinose, maltose, mannose,galactose, or oligosaccharides, directly or indirectly into the desiredfermentation product.

Examples of bacterial and fungal fermenting organisms producing ethanolare described by Lin et al., 2006, Appl. Microbiol. Biotechnol. 69:627-642.

Examples of fermenting microorganisms that can ferment C₆ sugars includebacterial and fungal organisms, such as yeast. Preferred yeast includesstrains of the Saccharomyces spp., preferably Saccharomyces cerevisiae.

Examples of fermenting organisms that can ferment C₅ sugars includebacterial and fungal organisms, such as some yeast. Preferred C₅fermenting yeast include strains of Pichia, preferably Pichia stipitis,such as Pichia stipitis CBS 5773; strains of Candida, preferably Candidaboidinii, Candida brassicae, Candida sheatae, Candida diddensii, Candidapseudotropicalis, or Candida utilis.

Other fermenting organisms include strains of Zymomonas, such asZymomonas mobilis; Hansenula, such as Hansenula anomala; Kluyveromyces,such as K. fragilis; Schizosaccharomyces, such as S. pombe; E. coli,especially E. coli strains that have been genetically modified toimprove the yield of ethanol; Clostridium, such as Clostridiumacetobutylicum, Chlostridium thermocellum, and Chlostridiumphytofermentans; Geobacillus sp.; Thermoanaerobacter, such asThermoanaerobacter saccharolyticum; and Bacillus, such as Bacilluscoagulans.

In a preferred aspect, the yeast is a Saccharomyces spp. In a morepreferred aspect, the yeast is Saccharomyces cerevisiae. In another morepreferred aspect, the yeast is Saccharomyces distaticus. In another morepreferred aspect, the yeast is Saccharomyces uvarum. In anotherpreferred aspect, the yeast is a Kluyveromyces. In another morepreferred aspect, the yeast is Kluyveromyces marxianus. In another morepreferred aspect, the yeast is Kluyveromyces fragilis. In anotherpreferred aspect, the yeast is a Candida. In another more preferredaspect, the yeast is Candida boidinii. In another more preferred aspect,the yeast is Candida brassicae. In another more preferred aspect, theyeast is Candida diddensii. In another more preferred aspect, the yeastis Candida pseudotropicalis. In another more preferred aspect, the yeastis Candida utilis. In another preferred aspect, the yeast is aClavispora. In another more preferred aspect, the yeast is Clavisporalusitaniae. In another more preferred aspect, the yeast is Clavisporaopuntiae. In another preferred aspect, the yeast is a Pachysolen. Inanother more preferred aspect, the yeast is Pachysolen tannophilus. Inanother preferred aspect, the yeast is a Pichia. In another morepreferred aspect, the yeast is a Pichia stipitis. In another preferredaspect, the yeast is a Bretannomyces. In another more preferred aspect,the yeast is Bretannomyces clausenii (Philippidis, G. P., 1996,Cellulose bioconversion technology, in Handbook on Bioethanol:Production and Utilization, Wyman, C. E., ed., Taylor & Francis,Washington, D.C., 179-212).

Bacteria that can efficiently ferment hexose and pentose to ethanolinclude, for example, Zymomonas mobilis, Clostridium acetobutylicum,Clostridium thermocellum, Chlostridium phytofermentans, Geobacillus sp.,Thermoanaerobacter saccharolyticum, and Bacillus coagulans (Philippidis,1996, supra).

In a preferred aspect, the bacterium is a Zymomonas. In a more preferredaspect, the bacterium is Zymomonas mobilis. In another preferred aspect,the bacterium is a Clostridium. In another more preferred aspect, thebacterium is Clostridium thermocellum.

Commercially available yeast suitable for ethanol production includes,e.g., ETHANOL RED™ yeast (Fermentis/Lesaffre, USA), FALI™ (Fleischmann'sYeast, USA), SUPERSTART™ and THERMOSACC™ fresh yeast (EthanolTechnology, WI, USA), BIOFERM™ AFT and XR (NABC-North AmericanBioproducts Corporation, GA, USA), GERT STRAND™ (Gert Strand AB,Sweden), and FERMIOL™ (DSM Specialties).

In a preferred aspect, the fermenting microorganism has been geneticallymodified to provide the ability to ferment pentose sugars, such asxylose utilizing, arabinose utilizing, and xylose and arabinoseco-utilizing microorganisms.

The cloning of heterologous genes into various fermenting microorganismshas led to the construction of organisms capable of converting hexosesand pentoses to ethanol (cofermentation) (Chen and Ho, 1993, Cloning andimproving the expression of Pichia stipitis xylose reductase gene inSaccharomyces cerevisiae, Appl. Biochem. Biotechnol. 39-40: 135-147; Hoet al., 1998, Genetically engineered Saccharomyces yeast capable ofeffectively cofermenting glucose and xylose, Appl. Environ. Microbiol.64: 1852-1859; Kotter and Ciriacy, 1993, Xylose fermentation bySaccharomyces cerevisiae, Appl. Microbiol. Biotechnol. 38: 776-783;Walfridsson et al., 1995, Xylose-metabolizing Saccharomyces cerevisiaestrains overexpressing the TKL1 and TALI genes encoding the pentosephosphate pathway enzymes transketolase and transaldolase, Appl.Environ. Microbiol. 61: 4184-4190; Kuyper et al., 2004, Minimalmetabolic engineering of Saccharomyces cerevisiae for efficientanaerobic xylose fermentation: a proof of principle, FEMS Yeast Research4: 655-664; Beall et al., 1991, Parametric studies of ethanol productionfrom xylose and other sugars by recombinant Escherichia coli, Biotech.Bioeng. 38: 296-303; Ingram et al., 1998, Metabolic engineering ofbacteria for ethanol production, Biotechnol. Bioeng. 58: 204-214; Zhanget al., 1995, Metabolic engineering of a pentose metabolism pathway inethanologenic Zymomonas mobilis, Science 267: 240-243; Deanda et al.,1996, Development of an arabinose-fermenting Zymomonas mobilis strain bymetabolic pathway engineering, Appl. Environ. Microbiol. 62: 4465-4470;WO 2003/062430, xylose isomerase).

In a preferred aspect, the genetically modified fermenting microorganismis Saccharomyces cerevisiae. In another preferred aspect, thegenetically modified fermenting microorganism is Zymomonas mobilis. Inanother preferred aspect, the genetically modified fermentingmicroorganism is Escherichia coli. In another preferred aspect, thegenetically modified fermenting microorganism is Klebsiella oxytoca. Inanother preferred aspect, the genetically modified fermentingmicroorganism is Kluyveromyces sp.

It is well known in the art that the organisms described above can alsobe used to produce other substances, as described herein.

The fermenting microorganism is typically added to the degradedlignocellulose or hydrolysate and the fermentation is performed forabout 8 to about 96 hours, such as about 24 to about 60 hours. Thetemperature is typically between about 26° C. to about 60° C., inparticular about 32° C. or 50° C., and at about pH 3 to about pH 8, suchas around pH 4-5, 6, or 7.

In a preferred aspect, the yeast and/or another microorganism is appliedto the degraded cellulosic material and the fermentation is performedfor about 12 to about 96 hours, such as typically 24-60 hours. In apreferred aspect, the temperature is preferably between about 20° C. toabout 60° C., more preferably about 25° C. to about 50° C., and mostpreferably about 32° C. to about 50° C., in particular about 32° C. or50° C., and the pH is generally from about pH 3 to about pH 7,preferably around pH 4-7. However, some fermenting organisms, e.g.,bacteria, have higher fermentation temperature optima. Yeast or anothermicroorganism is preferably applied in amounts of approximately 10⁵ to10¹², preferably from approximately 10⁷ to 10¹⁰, especiallyapproximately 2×10⁸ viable cell count per ml of fermentation broth.Further guidance in respect of using yeast for fermentation can be foundin, e.g., “The Alcohol Textbook” (Editors K. Jacques, T. P. Lyons and D.R. Kelsall, Nottingham University Press, United Kingdom 1999), which ishereby incorporated by reference.

For ethanol production, following the fermentation the fermented slurryis distilled to extract the ethanol. The ethanol obtained according tothe methods of the invention can be used as, e.g., fuel ethanol,drinking ethanol, i.e., potable neutral spirits, or industrial ethanol.

A fermentation stimulator can be used in combination with any of theprocesses described herein to further improve the fermentation process,and in particular, the performance of the fermenting microorganism, suchas, rate enhancement and ethanol yield. A “fermentation stimulator”refers to stimulators for growth of the fermenting microorganisms, inparticular, yeast. Preferred fermentation stimulators for growth includevitamins and minerals. Examples of vitamins include multivitamins,biotin, pantothenate, nicotinic acid, meso-inositol, thiamine,pyridoxine, para-aminobenzoic acid, folic acid, riboflavin, and VitaminsA, B, C, D, and E. See, for example, Alfenore et al., Improving ethanolproduction and viability of Saccharomyces cerevisiae by a vitaminfeeding strategy during fed-batch process, Springer-Verlag (2002), whichis hereby incorporated by reference. Examples of minerals includeminerals and mineral salts that can supply nutrients comprising P, K,Mg, S, Ca, Fe, Zn, Mn, and Cu.

Fermentation Products:

A fermentation product can be any substance derived from thefermentation. The fermentation product can be, without limitation, analcohol (e.g., arabinitol, butanol, ethanol, glycerol, methanol,1,3-propanediol, sorbitol, and xylitol); an organic acid (e.g., aceticacid, acetonic acid, adipic acid, ascorbic acid, citric acid,2,5-diketo-D-gluconic acid, formic acid, fumaric acid, glucaric acid,gluconic acid, glucuronic acid, glutaric acid, 3-hydroxypropionic acid,itaconic acid, lactic acid, malic acid, malonic acid, oxalic acid,oxaloacetic acid, propionic acid, succinic acid, and xylonic acid); aketone (e.g., acetone); an amino acid (e.g., aspartic acid, glutamicacid, glycine, lysine, serine, and threonine); and a gas (e.g., methane,hydrogen (H₂), carbon dioxide (CO₂), and carbon monoxide (CO)). Thefermentation product can also be protein as a high value product.

In a preferred aspect, the fermentation product is an alcohol. It willbe understood that the term “alcohol” encompasses a substance thatcontains one or more hydroxyl moieties. In a more preferred aspect, thealcohol is arabinitol. In another more preferred aspect, the alcohol isbutanol.

In another more preferred aspect, the alcohol is ethanol. In anothermore preferred aspect, the alcohol is glycerol. In another morepreferred aspect, the alcohol is methanol. In another more preferredaspect, the alcohol is 1,3-propanediol. In another more preferredaspect, the alcohol is sorbitol. In another more preferred aspect, thealcohol is xylitol. See, for example, Gong, C. S., Cao, N. J., Du, J.,and Tsao, G. T., 1999, Ethanol production from renewable resources, inAdvances in Biochemical Engineering/Biotechnology, Scheper, T., ed.,Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241; Silveira, M.M., and Jonas, R., 2002, The biotechnological production of sorbitol,Appl. Microbiol. Biotechnol. 59: 400-408; Nigam, P., and Singh, D.,1995, Processes for fermentative production of xylitol—a sugarsubstitute, Process Biochemistry 30 (2): 117-124; Ezeji, T. C., Qureshi,N. and Blaschek, H. P., 2003, Production of acetone, butanol and ethanolby Clostridium beijerinckii BA101 and in situ recovery by gas stripping,World Journal of Microbiology and Biotechnology 19 (6): 595-603.

In another preferred aspect, the fermentation product is an organicacid. In another more preferred aspect, the organic acid is acetic acid.In another more preferred aspect, the organic acid is acetonic acid. Inanother more preferred aspect, the organic acid is adipic acid. Inanother more preferred aspect, the organic acid is ascorbic acid. Inanother more preferred aspect, the organic acid is citric acid. Inanother more preferred aspect, the organic acid is 2,5-diketo-D-gluconicacid. In another more preferred aspect, the organic acid is formic acid.In another more preferred aspect, the organic acid is fumaric acid. Inanother more preferred aspect, the organic acid is glucaric acid. Inanother more preferred aspect, the organic acid is gluconic acid. Inanother more preferred aspect, the organic acid is glucuronic acid. Inanother more preferred aspect, the organic acid is glutaric acid. Inanother preferred aspect, the organic acid is 3-hydroxypropionic acid.In another more preferred aspect, the organic acid is itaconic acid. Inanother more preferred aspect, the organic acid is lactic acid. Inanother more preferred aspect, the organic acid is malic acid. Inanother more preferred aspect, the organic acid is malonic acid. Inanother more preferred aspect, the organic acid is oxalic acid. Inanother more preferred aspect, the organic acid is propionic acid. Inanother more preferred aspect, the organic acid is succinic acid. Inanother more preferred aspect, the organic acid is xylonic acid. See,for example, Chen, R., and Lee, Y. Y., 1997, Membrane-mediatedextractive fermentation for lactic acid production from cellulosicbiomass, Appl. Biochem. Biotechnol. 63-65: 435-448.

In another preferred aspect, the fermentation product is a ketone. Itwill be understood that the term “ketone” encompasses a substance thatcontains one or more ketone moieties. In another more preferred aspect,the ketone is acetone. See, for example, Qureshi and Blaschek, 2003,supra.

In another preferred aspect, the fermentation product is an amino acid.In another more preferred aspect, the organic acid is aspartic acid. Inanother more preferred aspect, the amino acid is glutamic acid. Inanother more preferred aspect, the amino acid is glycine. In anothermore preferred aspect, the amino acid is lysine. In another morepreferred aspect, the amino acid is serine. In another more preferredaspect, the amino acid is threonine. See, for example, Richard, A., andMargaritis, A., 2004, Empirical modeling of batch fermentation kineticsfor poly(glutamic acid) production and other microbial biopolymers,Biotechnology and Bioengineering 87 (4): 501-515.

In another preferred aspect, the fermentation product is a gas. Inanother more preferred aspect, the gas is methane. In another morepreferred aspect, the gas is H₂. In another more preferred aspect, thegas is CO₂. In another more preferred aspect, the gas is CO. See, forexample, Kataoka, N., A. Miya, and K. Kiriyama, 1997, Studies onhydrogen production by continuous culture system of hydrogen-producinganaerobic bacteria, Water Science and Technology 36 (6-7): 41-47; andGunaseelan V. N. in Biomass and Bioenergy, Vol. 13 (1-2), pp. 83-114,1997, Anaerobic digestion of biomass for methane production: A review.

Recovery.

The fermentation product(s) can be optionally recovered from thefermentation medium using any method known in the art including, but notlimited to, chromatography, electrophoretic procedures, differentialsolubility, distillation, or extraction. For example, alcohol isseparated from the fermented cellulosic material and purified byconventional methods of distillation. Ethanol with a purity of up toabout 96 vol. % can be obtained, which can be used as, for example, fuelethanol, drinking ethanol, i.e., potable neutral spirits, or industrialethanol.

Plants

The present invention also relates to plants, e.g., a transgenic plant,plant part, or plant cell, comprising a polynucleotide of the presentinvention so as to express and produce the variant in recoverablequantities. The variant may be recovered from the plant or plant part.Alternatively, the plant or plant part containing the variant may beused as such for improving the quality of a food or feed, e.g.,improving nutritional value, palatability, and rheological properties,or to destroy an antinutritive factor.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot). Examples of monocot plants are grasses, such as meadowgrass (blue grass, Poa), forage grass such as Festuca, Lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape seed, and the closely relatedmodel organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems.Specific plant cell compartments, such as chloroplasts, apoplasts,mitochondria, vacuoles, peroxisomes and cytoplasm are also considered tobe a plant part. Furthermore, any plant cell, whatever the tissueorigin, is considered to be a plant part. Likewise, plant parts such asspecific tissues and cells isolated to facilitate the utilization of theinvention are also considered plant parts, e.g., embryos, endosperms,aleurone and seed coats.

Also included within the scope of the present invention are the progenyof such plants, plant parts, and plant cells.

The transgenic plant or plant cell expressing a variant may beconstructed in accordance with methods known in the art. In short, theplant or plant cell is constructed by incorporating one or more(several) expression constructs encoding a variant into the plant hostgenome or chloroplast genome and propagating the resulting modifiedplant or plant cell into a transgenic plant or plant cell.

The expression construct is conveniently a nucleic acid construct thatcomprises a polynucleotide encoding a variant operably linked withappropriate regulatory sequences required for expression of thepolynucleotide in the plant or plant part of choice. Furthermore, theexpression construct may comprise a selectable marker useful foridentifying plant cells into which the expression construct has beenintegrated and DNA sequences necessary for introduction of the constructinto the plant in question (the latter depends on the DNA introductionmethod to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences, is determined, forexample, on the basis of when, where, and how the variant is desired tobe expressed. For instance, the expression of the gene encoding avariant may be constitutive or inducible, or may be developmental, stageor tissue specific, and the gene product may be targeted to a specifictissue or plant part such as seeds or leaves. Regulatory sequences are,for example, described by Tague et al., 1988, Plant Physiol. 86: 506.

For constitutive expression, the 35S-CaMV, the maize ubiquitin 1, andthe rice actin 1 promoter may be used (Franck et al., 1980, Cell 21:285-294; Christensen et al., 1992, Plant Mol. Biol. 18: 675-689; Zhanget al., 1991, Plant Cell 3: 1155-1165). Organ-specific promoters may be,for example, a promoter from storage sink tissues such as seeds, potatotubers, and fruits (Edwards and Coruzzi, 1990, Ann. Rev. Genet. 24:275-303), or from metabolic sink tissues such as meristems (Ito et al.,1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such asthe glutelin, prolamin, globulin, or albumin promoter from rice (Wu etal., 1998, Plant Cell Physiol. 39: 885-889), a Vicia faba promoter fromthe legumin B4 and the unknown seed protein gene from Vicia faba (Conradet al., 1998, J. Plant Physiol. 152: 708-711), a promoter from a seedoil body protein (Chen et al., 1998, Plant Cell Physiol. 39: 935-941),the storage protein napA promoter from Brassica napus, or any other seedspecific promoter known in the art, e.g., as described in WO 91/14772.Furthermore, the promoter may be a leaf specific promoter such as therbcs promoter from rice or tomato (Kyozuka et al., 1993, Plant Physiol.102: 991-1000), the chlorella virus adenine methyltransferase genepromoter (Mitra and Higgins, 1994, Plant Mol. Biol. 26: 85-93), the aldPgene promoter from rice (Kagaya et al., 1995, Mol. Gen. Genet. 248:668-674), or a wound inducible promoter such as the potato pin2 promoter(Xu et al., 1993, Plant Mol. Biol. 22: 573-588). Likewise, the promotermay inducible by abiotic treatments such as temperature, drought, oralterations in salinity or induced by exogenously applied substancesthat activate the promoter, e.g., ethanol, oestrogens, plant hormonessuch as ethylene, abscisic acid, and gibberellic acid, and heavy metals.

A promoter enhancer element may also be used to achieve higherexpression of a variant in the plant. For instance, the promoterenhancer element may be an intron that is placed between the promoterand the polynucleotide encoding a variant. For instance, Xu et al.,1993, supra, disclose the use of the first intron of the rice actin 1gene to enhance expression.

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Presently, Agrobacterium tumefaciens-mediated gene transfer is themethod of choice for generating transgenic dicots (for a review, seeHooykas and Schilperoort, 1992, Plant Mol. Biol. 19: 15-38) and can alsobe used for transforming monocots, although other transformation methodsare often used for these plants. Presently, the method of choice forgenerating transgenic monocots is particle bombardment (microscopic goldor tungsten particles coated with the transforming DNA) of embryoniccalli or developing embryos (Christou, 1992, Plant J. 2: 275-281;Shimamoto, 1994, Curr. Opin. Biotechnol. 5: 158-162; Vasil et al., 1992,Bio/Technology 10: 667-674). An alternative method for transformation ofmonocots is based on protoplast transformation as described by Omirullehet al., 1993, Plant Mol. Biol. 21: 415-428. Additional transformationmethods for use in accordance with the present disclosure include thosedescribed in U.S. Pat. Nos. 6,395,966 and 7,151,204 (both of which areherein incorporated by reference in their entirety).

Following transformation, the transformants having incorporated theexpression construct are selected and regenerated into whole plantsaccording to methods well known in the art. Often the transformationprocedure is designed for the selective elimination of selection geneseither during regeneration or in the following generations by using, forexample, co-transformation with two separate T-DNA constructs or sitespecific excision of the selection gene by a specific recombinase.

In addition to direct transformation of a particular plant genotype witha construct prepared according to the present invention, transgenicplants may be made by crossing a plant having the construct to a secondplant lacking the construct. For example, a construct encoding a variantcan be introduced into a particular plant variety by crossing, withoutthe need for ever directly transforming a plant of that given variety.Therefore, the present invention encompasses not only a plant directlyregenerated from cells which have been transformed in accordance withthe present invention, but also the progeny of such plants. As usedherein, progeny may refer to the offspring of any generation of a parentplant prepared in accordance with the present invention. Such progenymay include a DNA construct prepared in accordance with the presentinvention, or a portion of a DNA construct prepared in accordance withthe present invention. Crossing results in the introduction of atransgene into a plant line by cross pollinating a starting line with adonor plant line. Non-limiting examples of such steps are furtherarticulated in U.S. Pat. No. 7,151,204.

Plants may be generated through a process of backcross conversion. Forexample, plants include plants referred to as a backcross convertedgenotype, line, inbred, or hybrid.

Genetic markers may be used to assist in the introgression of one ormore transgenes of the invention from one genetic background intoanother. Marker assisted selection offers advantages relative toconventional breeding in that it can be used to avoid errors caused byphenotypic variations. Further, genetic markers may provide dataregarding the relative degree of elite germplasm in the individualprogeny of a particular cross. For example, when a plant with a desiredtrait which otherwise has a non-agronomically desirable geneticbackground is crossed to an elite parent, genetic markers may be used toselect progeny which not only possess the trait of interest, but alsohave a relatively large proportion of the desired germplasm. In thisway, the number of generations required to introgress one or more traitsinto a particular genetic background is minimized.

The present invention also relates to methods of producing a variant ofthe present invention comprising: (a) cultivating a transgenic plant ora plant cell comprising a polynucleotide encoding the variant underconditions conducive for production of the variant; and (b) recoveringthe variant.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES

Materials

Chemicals used as buffers and substrates were commercial products of atleast reagent grade.

Strains

Thielavia terrestris NRRL 8126 was used as the source of DNA encodingthe Family 6A cellobiohydrolase II. Aspergillus oryzae JaL250 strain (WO99/61651) was used for expression of the Thielavia terrestriscellobiohydrolase II.

Media

PDA plates were composed of 39 grams of potato dextrose agar anddeionized water to 1 liter.

MDU2BP medium was composed of 45 g of maltose, 1 g of MgSO₄.7H₂O, 1 g ofNaCl, 2 g of K₂SO₄, 12 g of KH2PO4, 7 g of yeast extract, 2 g of urea,0.5 ml of AMG trace metals solution, and deionized water to 1 liter, pHto 5.0.

AMG trace metals solution was composed of 14.3 g of ZnSO₄.7H₂O, 2.5 g ofCuSO₄.5H₂O, 0.5 g of NiCl₂.6H₂O, 13.8 g of FeSO₄.7H₂O, 8.5 g ofMnSO₄.H₂O, 3 g of citric acid, and deionized water to 1 liter.

Example 1: Construction of a Cloning Vector for the Thielavia terrestrisFamily GH6A Cellobiohydrolase II Gene

Two synthetic oligonucleotide primers shown below were designed to PCRamplify a polynucleotide encoding the Thielavia terrestris Family GH6Acellobiohydrolase II from cDNA clone Tter11C₉ containing pTter11C₉described in U.S. Pat. No. 7,220,565 (SEQ ID NO: 1 for the cDNA sequenceand SEQ ID NO: 2 for the deduced amino acid sequence).

Forward primer: (SEQ ID NO: 133) 5′-ACTGGATTTACCatggctcag-3′Reverse primer: (SEQ ID NO: 134) 5′-TCACCTCTAGTTAATTAActaaaagggcggg-3′

A total of 37.5 picomoles of each of the primers above were used in aPCR reaction containing 40 ng of pTter11C9, 1×Pfx Amplification Buffer(Invitrogen, Carlsbad, Calif., USA), 1.5 μl of a blend of dATP, dTTP,dGTP, and dCTP, each at 10 mM, 1.25 units of PLATINUM® Pfx DNAPolymerase (Invitrogen, Carlsbad, Calif., USA), and 1 μl of 50 mM MgSO₄,in a final volume of 50 μl. The amplification reaction was performed ina PTC-200 DNA Engine® thermocycler (MJ Research, Inc., Waltham, Mass.,USA) programmed for one cycle at 95° C. for 30 seconds; and 30 cycleseach at 95° C. for 15 seconds, 55° C. for 30 seconds, and 68° C. for 1.5minutes. After the 30 cycles, the reaction was heated for 10 minutes at68° C. The heat block then went to a 4° C. soak cycle.

The reaction products were isolated by 1.0% agarose gel electrophoresisusing 40 mM Tris base-20 mM sodium acetate-1 mM disodium EDTA (TAE)buffer where a 1.5 kb product band was excised from the gel andextracted using a QIAQUICK® Gel Extraction Kit (QIAGEN Inc., Valencia,Calif., USA) according to the manufacturer's instructions.

The purified 1.5 kb PCR product was inserted into pCR®2.1-TOPO® using aTOPO® TA Cloning Kit (Invitrogen, Carlsbad, Calif., USA). Overhangs of3′ adenine were added by mixing 1 μl of 10× ThermoPol buffer (NewEngland Biolabs, Inc., Ipswich, Mass., USA), 4 μl of gel purified PCRproduct, 4 μl of water, 0.5 μl of 10 mM dNTPs, and 0.5 μl of Taq DNApolymerase (Invitrogen, Carlsbad, Calif., USA) and incubating for 10minutes at 72° C. Two microliters of the reaction were then mixed with 2μl of water, 1 μl of 1.2 M NaCl, and 1 μl of pCR®2.1 TOPO® mix andincubated at room temperature for 5 minutes. E. coli ONE SHOT® TOP10cells (Invitrogen, Carlsbad, Calif., USA) were transformed with 2 μl ofthis mixture according to the manufacturer's instructions. Plasmid DNAfrom several of the resulting E. coli transformants was prepared using aBIOROBOT® 9600 (QIAGEN Inc., Valencia, Calif., USA). A plasmidcontaining a polynucleotide encoding the Thielavia terrestris FamilyGH6A cellobiohydrolase II was identified and the full gene sequence wasdetermined using a 3130xl Genetic Analyzer (Applied Biosystems, FosterCity, Calif., USA).

An internal Nco I restriction site was removed by performingsite-directed mutagenesis using a QUIKCHANGE® XL Site-DirectedMutagenesis Kit (Stratagene, La Jolla, Calif., USA) according to themanufacturer's instructions and the two synthetic oligonucleotideprimers shown below:

Forward primer: (SEQ ID NO: 135)5′-cccagcatgacgggcgcaatggccaccaaggcggcc-3′ Reverse primer:(SEQ ID NO: 136) 5′-ggccgccttggtggccattgcgcccgtcatgctggg-3

The resulting pMaWo1 plasmid DNA was prepared using a BIOROBOT® 9600.Plasmid pMaWo1 was sequenced using a 3130xl Genetic Analyzer.

Example 2: Construction of the Thielavia terrestris Family GH6ACellobiohydrolase II Gene Variants

Variants of the Thielavia terrestris GH6A cellobiohydrolase II wereconstructed by performing site-directed mutagenesis on pMaWo1 using aQUIKCHANGE® XL Site-Directed Mutagenesis Kit. A summary of the oligosused for the site-directed mutagenesis and the variants obtained areshown in Table 1.

The resulting variant plasmid DNAs were prepared using a BIOROBOT® 9600.Variant plasmid constructs were sequenced using a 3130xl GeneticAnalyzer.

TABLE 1 Cloning Amino acid Primer Plasmid changes name Sequences NameA272S MaWo64 gaacgtggccaagtgc pMaWo17 tccaacgccgagtcga c(SEQ ID NO: 137) MaWo65 gtcgactcggcgttgg agcacttggccacgtt c(SEQ ID NO: 138) Q287K MaWo31 gaccgtctacgcgctg pMaWo11 aagcagctgaacctg(SEQ ID NO: 139) MaWo32 caggttcagctgcttc agcgcgtagacggtc(SEQ ID NO: 140) S325D MaWo94 gccgagatctacacgg pMaWo29 acgccggcaagccgg(SEQ ID NO: 141) MaWo95 ccggcttgccggcgtc cgtgtagatctcggc(SEQ ID NO: 142) L347I MaWo21 caactacaacggctgg pMaWo6 agcatagctacgccgccctcgtacacc (SEQ ID NO: 143) MaWo22 ggtgtacgagggcggc gtagctatgctccagccgttgtagttg (SEQ ID NO: 144) D357N MaWo19 gccctcgtacacccag pMaWo5ggtaaccccaactacg acgagagc (SEQ ID NO: 145) MaWo20 gctctcgtcgtagttggggttaccctgggtgt acgagggc (SEQ ID NO: 146) S363K MaWo27 gaccccaactacgacgpMaWo10 agaagcactacgtcca ggccc (SEQ ID NO: 147) MaWo28 gggcctggacgtagtgcttctcgtcgtagttg gggtc (SEQ ID NO: 148) C435S MaWo6 caagcccggcggcgagpMaWo3 tccgacggcacgagca ac (SEQ ID NO: 149) MaWo7 gttgctcgtgccgtcggactcgccgccgggct tg (SEQ ID NO: 150) T464Q MaWo37 gcagcctgctccggagpMaWo14 gctggccaatggttcc aggcctacttcg (SEQ ID NO: 151) MaWo38cgaagtaggcctggaa ccattggccagcctcc ggagcaggctgc (SEQ ID NO: 152) G409CMaWo142 caacgttatcggaact pMaWo48i tgcttcggcgtgcgcc (SEQ ID NO: 153)MaWo143 ggcgcacgccgaagca agttccgataacgttg (SEQ ID NO: 154) G409C +MaWo144 cgagcagctcctgacc pMaWo48* N476C tgcgccaacccgccct tttag(SEQ ID NO: 155) MaWo145 ctaaaagggcgggttg gcgcaggtcaggagct gctcg(SEQ ID NO: 156) *Plasmid pMaWo48 comprises both G409C + N476C.

Example 3: Construction of an Aspergillus oryzae Expression Vector forthe Thielavia terrestris Family GH6A Cellobiohydrolase II Variants

Two synthetic oligonucleotide primers shown below were designed to PCRamplify the cDNAs encoding the Thielavia terrestris Family GH6Acellobiohydrolase II variants from pMaWo3, pMaWo5, pMaWo6, pMaWo10,pMaWo11, pMaWo14, pMaWo17, pMaWo29, and pMaWo48.

Forward primer: (SEQ ID NO: 157) 5′-ACTGGATTTACCATGGCTCAG-3′Reverse primer: (SEQ ID NO: 158) 5′-TCACCTCTAGTTAATTAAGTAAAAGGGCGGG-3′

Bold letters represent coding sequence. The remaining sequence ishomologous to the insertion sites of pAlLo2 (WO 2005/074647).

The amplification reactions were each composed of 37.5 picomoles of eachof the primers above, 40 ng of pMaWo3, pMaWo5, pMaWo6, pMaWo10, pMaWo11,pMaWo14, pMaWo17, pMaWo29, or pMaWo48, 1×Pfx Amplification Buffer, 1.5μl of a blend of dATP, dTTP, dGTP, and dCTP, each at 10 mM, 1.25 unitsof PLATINUM® Pfx DNA Polymerase, and 1 μl of 50 mM MgSO₄, in a finalvolume of 50 μl. The amplifications were performed using an EPPENDORF®MASTERCYCLER® ep gradient S thermocycler (Eppendorf Scientific, Inc.,Westbury, N.Y., USA) programmed for one cycle at 95° C. for 30 seconds;and 30 cycles each at 95° C. for 15 seconds, 55° C. for 30 seconds, and68° C. for 1.5 minutes. After the 30 cycles, the reactions were heatedfor 10 minutes at 68° C. The heat block then went to a 4° C. soak cycle.

Each of the reaction products were isolated by 1.0% agarose gelelectrophoresis using TAE buffer where a 1.5 kb product band for eachamplification was excised from the gels and extracted using a QIAQUICK®Gel Extraction Kit.

An IN-FUSION® Cloning Kit (BD Biosciences, Palo Alto, Calif., USA) wasused to clone each of the fragments directly into the expression vectorpAlLo2, without the need for restriction digests and ligation. Thevector was digested with Nco I and Pac I. Each of the fragments waspurified by gel electrophoresis described above. The digested vector wascombined with each of the fragments in reactions resulting in expressionplasmids under which transcription of the Family GH6A cellobiohydrolaseII cDNA and mutants thereof were under the control of the NA2-tpipromoter (a modified promoter from the gene encoding neutralalpha-amylase in Aspergillus niger in which the untranslated leader hasbeen replaced by an untranslated leader from the gene encoding triosephosphate isomerase in Aspergillus nidulans). The recombinationreactions (20 μl) were composed of 1×IN-FUSION® Buffer (BD Biosciences,Palo Alto, Calif., USA), 1×BSA (BD Biosciences, Palo Alto, Calif., USA),1 μl of IN-FUSION® enzyme (diluted 1:10) (BD Biosciences, Palo Alto,Calif., USA), 160 ng of pAlLo2 digested with NcoI and PacI, and 100 ngof each of the Thielavia terrestris GH6A cellobiohydrolase II purifiedPCR products. The reactions were incubated at room temperature for 30minutes. One μl of each reaction was used to transform E. coli ONE SHOT®TOP10 cells. Plasmid DNA from the E. coli transformants containingpMaWo3EV2 (C435S), pMaWo5EV2 (D357N), pMaWo6EV2 (L347I), pMaWo1OEV2(S363K), pMaWo11EV2 (Q287K), pMaWo14EV2 (T464Q), pMaWo17EV2 (A272S),pMaWo29EV2 (S325D), or pMaWo48EV2 (G409C+N476C) was prepared using aBIOROBOT® 9600. Plasmids were sequenced using a 3130xl Genetic Analyzer.

Example 4: Expression of the Thielavia terrestris cDNA Encoding FamilyGH6A Cellobiohydrolase II Variants in Aspergillus oryzae JaL250

Aspergillus oryzae JaL250 protoplasts were prepared according to themethod of Christensen et al., 1988, Bio/Technology 6: 1419-1422 andtransformed with 5 μg of expression vector (pMaWo3EV2, pMaWo5EV2,pMaWo6EV2, pMaWo10EV2, pMaWo11EV2, pMaWo14EV2, pMaWo17EV2, pMaWo29EV2,or pMaWo48EV2). Expression vector pAlLo21 (U.S. Pat. No. 7,220,565) wastransformed into Aspergillus oryzae JaL250 for expression of theThielavia terrestris Family GH6A wild-type cellobiohydrolase II gene.

The transformation of Aspergillus oryzae JaL250 with pAlLo21, pMaWo3EV2,pMaWo5EV2, pMaWo6EV2, pMaWo10EV2, pMaWo11EV2, pMaWo14EV2, pMaWo17EV2,pMaWo29EV2, or pMaWo48EV2 yielded about 1-10 transformants for eachvector. Up to four transformants for each transformation were isolatedto individual PDA plates.

Confluent PDA plates of the transformants were washed with 8 ml of 0.01%TWEEN® 20 and inoculated separately into 1 ml of MDU2BP medium insterile 24 well tissue culture plates and incubated at 34° C. Three daysafter incubation, 20 μl of harvested broth from each culture wereanalyzed using 8-16% Tris-Glycine SDS-PAGE gels (Invitrogen, Carlsbad,Calif., USA) according to the manufacturer's instructions. SDS-PAGEprofiles of the cultures showed that several transformants had a newmajor band of approximately 75 kDa.

A confluent plate of one transformant for each transformation (grown ona PDA plate) was washed with 8 ml of 0.01% TWEEN® 20 and inoculated into500 ml glass shake flasks containing 100 ml of MDU2BP medium andincubated at 34° C., 200 rpm to generate broth for characterization ofthe enzyme. The flasks were harvested on day 3 and filtered using a 0.22μm GP Express plus Membrane (Millipore, Bedford, Mass., USA).

Wild-type Thielavia terrestris cellobiohydrolase II was produced usingpAlLo21 according to WO 2006/074435.

Example 5: Measuring Thermostability of Thielavia terrestris Family GH6ACellobiohydrolase II Variants

Three ml of filtered broth for each culture from Example 4 were desaltedinto 100 mM NaCl-50 mM sodium acetate pH 5.0 using ECONO-PAC® 10DGDesalting Columns (Bio-Rad Laboratories, Inc., Hercules, Calif., USA).Protein in each desalted broth was concentrated into a 0.5 ml volumeusing a VIVASPIN® 6 Centrifugal Concentrator, 5 kDa molecular weightcut-off ultrafilter (Argos Technologies, Inc., Elgin, Ill., USA).

Concentrated broths were diluted to 1 mg/ml protein concentration using100 mM NaCl-50 mM sodium acetate pH 5.0. Two 25 μl aliquots of each 1mg/ml protein sample were added to THERMOWELL® tube strip PCR tubes(Corning, Corning, N.Y., USA). One aliquot was kept on ice while theother aliquot was heated in an EPPENDORF® MASTERCYCLER® ep gradient Sthermocycler for 20 minutes at 67° C. and then cooled to 4° C. beforebeing put on ice. Both samples were then diluted with 175 μl of 100 mMNaCl-50 mM sodium acetate pH 5.0.

Residual activity of the heated sample was then measured by determiningthe activity of the heated sample and the sample kept on ice inhydrolysis of phosphoric acid swollen cellulose (PASC). Ten microlitersof each sample was added in triplicate to a 96 well PCR plate(Eppendorf, Westbury, N.Y., USA). Then 190 μl of 2.1 g/I PASC was addedto the 10 μl of sample and mixed. Glucose standards at 100, 75, 50, 25,12.5 and 0 mg per liter in 50 mM sodium acetate pH 5.0 buffer were addedin duplicate at 200 μl per well. The resulting mixture was incubated for30 minutes at 50° C. in an EPPENDORF® MASTERCYCLER® ep gradient Sthermocycler. The reaction was stopped by addition of 50 μl of 0.5 MNaOH to each well, including the glucose standards. The plate was thencentrifuged in a Sorvall RT 6000D centrifuge (Thermo Scientific,Waltham, Mass., USA) with a Sorvall 1000B rotor equipped with amicroplate carrier (Thermo Scientific, Waltham, Mass., USA) for 2minutes at 2,000 rpm.

Activity on PASC was determined by measuring reducing ends releasedduring a 30 minute hydrolysis at 50° C. One hundred microliters ofsupernatant from the spun plate was transferred to a separate 96-wellPCR plate. Fifty microliters of 1.5% (w/v) PHBAH (4-hydroxy-benzhydride,Sigma Chemical Co., St. Louis, Mo., USA) in 0.5 M NaOH were added toeach well. The plate was then heated in an EPPENDORF® MASTERCYCLER® epgradient S thermocycler at 95° C. for 15 minutes and then 15° C. for 5minutes. A total of 100 μl of each sample was transferred to a clear,flat-bottom 96-well plate (Corning, Inc., Oneonta, N.Y., USA;). Theabsorbance at 410 nm was then measured using a SPECTRAMAX® 340pcspectrophotometric plate reader (Molecular Devices, Sunnyvale, Calif.,USA). The concentration of reducing ends released was determined from astraight-line fit to the concentration of reducing ends released versusthe absorbance at 410 nm for glucose standards. Residual activity wasthen calculated by dividing the reducing ends released from PASChydrolyzed by the heated sample by the reducing ends released from PASChydrolyzed by the sample that was kept on ice. Activity of thecellobiohydrolase II variants was compared to activity of the wild-typeprotein.

The results shown in FIG. 1 demonstrated an increase in thermostabilityby a higher residual activity for each variant compared to the wild-typeprotein.

Deposit of Biological Material

The following biological material has been deposited under the terms ofthe Budapest Treaty with the Agricultural Research Service PatentCulture Collection (NRRL), Northern Regional Research Center, 1815University Street, Peoria, Ill., 61604, USA, and given the followingaccession number:

Deposit Accession Number Date of Deposit E. coli pTter6A NRRL B-30802Dec. 17, 2004

The strain has been deposited under conditions that assure that accessto the culture will be available during the pendency of this patentapplication to one determined by foreign patent laws to be entitledthereto. The deposit represents a substantially pure culture of thedeposited strain. The deposit is available as required by foreign patentlaws in countries wherein counterparts of the subject application, orits progeny are filed. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentalaction.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

1-27. (canceled)
 28. An isolated variant of a parent cellobiohydrolaseII, comprising a substitution at a position corresponding to position325 of SEQ ID NO: 2, wherein the variant has cellobiohydrolase IIactivity and is selected from the group consisting of: (a) a varianthaving at least 95% sequence identity to residues 18-481 of SEQ ID NO:2; (b) a variant encoded by a polynucleotide that hybridizes under veryhigh stringency conditions with (i) nucleotides 52-1443 of SEQ ID NO: 1,or (ii) the full-length complement of (i), wherein very high stringencyconditions are defined as prehybridization and hybridization at 42° C.in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmonsperm DNA, and 50% formamide, and washing three times each for 15minutes using 2×SSC, 0.2% SDS at 70° C.; and (c) a variant encoded by apolynucleotide having at least 95% sequence identity to nucleotides52-1443 of SEQ ID NO: 1 or the genomic DNA sequence thereof.
 29. Thevariant of claim 28, which comprises a substitution at a positioncorresponding to position 325 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp,Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, orTyr.
 30. The variant of claim 29, wherein the substitution is Asp. 31.The variant of claim 28, wherein the variant has at least 96% sequenceidentity to residues 18-481 of SEQ ID NO:
 2. 32. The variant of claim28, wherein the variant has at least 97% sequence identity to residues18-481 of SEQ ID NO:
 2. 33. The variant of claim 28, wherein the varianthas at least 98% sequence identity to residues 18-481 of SEQ ID NO: 2.34. The variant of claim 28, wherein the variant has at least 99%sequence identity to residues 18-481 of SEQ ID NO:
 2. 35. The variant ofclaim 28, wherein the parent cellobiohydrolase II is selected from thegroup consisting of: (a) a polypeptide having at least 95% sequenceidentity to residues 18-481 of SEQ ID NO: 2; (b) a polypeptide encodedby a polynucleotide that hybridizes under very high stringencyconditions with (i) nucleotides 52-1443 of SEQ ID NO: 1, or (ii) thefull-length complement of (i), wherein very high stringency conditionsare defined as prehybridization and hybridization at 42° C. in 5×SSPE,0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and50% formamide, and washing three times each for 15 minutes using 2×SSC,0.2% SDS at 70° C.; and (c) a polypeptide encoded by a polynucleotidehaving at least 95% sequence identity to nucleotides 52-1443 of SEQ IDNO:
 1. 36. The variant of claim 28, wherein the parent cellobiohydrolaseII has at least 96% sequence identity to residues 18-481 of SEQ ID NO:2.
 37. The variant of claim 28, wherein the parent cellobiohydrolase IIhas at least 97% sequence identity to residues 18-481 of SEQ ID NO: 2.38. The variant of claim 28, wherein the parent cellobiohydrolase II hasat least 98% sequence identity to residues 18-481 of SEQ ID NO:
 2. 39.The variant of claim 28, wherein the parent cellobiohydrolase II has atleast 99% sequence identity to residues 18-481 of SEQ ID NO:
 2. 40. Thevariant of claim 28, wherein the parent cellobiohydrolase II comprisesresidues 18-481 of SEQ ID NO: 2, or a fragment thereof havingcellobiohydrolase activity.
 41. The variant of claim 28, wherein theparent cellobiohydrolase II comprises residues 18-481 of SEQ ID NO: 2.42. The variant of claim 28, wherein the variant has increasedthermostability relative to the parent.
 43. An isolated polynucleotideencoding the variant of claim
 28. 44. A recombinant host cell comprisingthe isolated polynucleotide of claim
 43. 45. A method of producing avariant of a parent cellobiohydrolase II, the method comprising: (a)cultivating an isolated host cell comprising the isolated polynucleotideof claim 43 under conditions suitable for the expression of the variant;and (b) recovering the variant.
 46. A transgenic plant, plant part orplant cell transformed with the isolated polynucleotide of claim
 43. 47.A method of producing the variant of claim 28, the method comprising:(a) cultivating a transgenic plant or a plant cell comprising apolynucleotide encoding the variant under conditions conducive forproduction of the variant; and (b) recovering the variant.
 48. A methodfor degrading a cellulosic material, comprising: treating the cellulosicmaterial with an enzyme composition comprising the variant of claim 28.49. The method of claim 48, further comprising recovering the degradedcellulosic material.
 50. A method for producing a fermentation product,comprising: (a) saccharifying a cellulosic material with an enzymecomposition comprising the variant of claim 28; (b) fermenting thesaccharified cellulosic material with one or more fermentingmicroorganisms to produce the fermentation product; and (c) recoveringthe fermentation product from the fermentation.
 51. A method offermenting a cellulosic material, comprising: fermenting the cellulosicmaterial with one or more fermenting microorganisms, wherein thecellulosic material is saccharified with an enzyme compositioncomprising the variant of claim
 28. 52. The method of claim 51, whereinthe fermenting of the cellulosic material produces a fermentationproduct.
 53. The method of claim 52, further comprising recovering thefermentation product from the fermentation.